| 质粒名称: | pNeae2 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 168300 |
| 存储实验室: | Luis Ángel Fernández |
|
载体骨架基本信息 |
|
| 载体名称: | pAK-Not |
| 载体出品公司: | Luis Angel Fernández |
| 空载体大小: | 3735 bp |
| 完整质粒大小: | 5832 bp |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 载体抗性: | 氯霉素, 25 μg/mL |
| 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: | Enterohemorraghic E. coli Intimin N-terminal fragment, E-tag, 6xHis tag, and myc-tag at the C-terminus |
| 物种来源: | Synthetic |
| 插入大小: | 2097 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | LacI-Plac |
| 标签/融合蛋白: |
E-tag（插入件上的C端子）
6xHis（插入件上的C端子）
myc-tag（插入件上的C端子）
|
| 克隆方法: | 限制性内切酶 |
| 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: | pNeae2 was a gift from Luis Ángel Fernández (Addgene plasmid # 168300 ; http://n2t.net/addgene:168300 ; RRID:Addgene_168300) |
| 参考文献部分: | Selection of single domain antibodies from immune libraries displayed on the surface of E. coli cells with two beta-domains of opposite topologies. Salema V, Marin E, Martinez-Arteaga R, Ruano-Gallego D, Fraile S, Margolles Y, Teira X, Gutierrez C, Bodelon G, Fernandez LA. PLoS One. 2013 Sep 23;8(9):e75126. doi: 10.1371/journal.pone.0075126. eCollection 2013. 10.1371/journal.pone.0075126 PubMed 24086454 |
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 pNeae2 5832 bp ds-DNA circular SYN 09-JUN-2021
DEFINITION Bacterial surface display of polypeptides (nanobodies) in E. coli.
ACCESSION .
VERSION .
KEYWORDS pNeae2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5832)
AUTHORS Salema V, Marin E, Martinez-Arteaga R, Ruano-Gallego D, Fraile S,
Margolles Y, Teira X, Gutierrez C, Bodelon G, Fernandez LA
TITLE Selection of single domain antibodies from immune libraries
displayed on the surface of E. coli cells with two beta-domains of
opposite topologies.
JOURNAL PLoS One. 2013 Sep 23;8(9):e75126. doi:
10.1371/journal.pone.0075126. eCollection 2013.
PUBMED 24086454
REFERENCE 2 (bases 1 to 5832)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jun 9, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..5832
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 1..31
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 39..55
/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 44..66
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 63..79
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 63..79
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
CDS 2131..2148
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
rep_origin 2283..2738
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2370..2389)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2580..2601
/label=F1ori-F
/note="F1 origin, forward primer"
CDS 2925..3584
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
primer_bind complement(2991..3010)
/label=CAT-R
/note="Chloramphenicol resistance gene, reverse primer"
rep_origin 3916..4504
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4405..4424
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
promoter 4569..4646
/gene="lacI"
/label=lacI promoter
CDS 4647..5729
/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(4666..4685)
/label=LacI-R
/note="LacI, reverse primer"
protein_bind 5797..5818
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
ORIGIN
1 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
61 cacaggaaac agctatgacc atgattacga atttctagat aacgagggca aatcatgatt
121 actcatggtt gttatacccg gacccggcac aagcataagc taaaaaaaac attgattatg
181 cttagtgctg gtttaggatt gtttttttat gttaatcaga attcatttgc aaatggtgaa
241 aattatttta aattgggttc ggattcaaaa ctgttaactc atgatagcta tcagaatcgc
301 cttttttata cgttgaaaac tggtgaaact gttgccgatc tttctaaatc gcaagatatt
361 aatttatcga cgatttggtc gttgaataag catttataca gttctgaaag cgaaatgatg
421 aaggccgcgc ctggtcagca gatcattttg ccactcaaaa aacttccctt tgaatacagt
481 gcactaccac ttttaggttc ggcacctctt gttgctgcag gtggtgttgc tggtcacacg
541 aataaactga ctaaaatgtc cccggacgtg accaaaagca acatgaccga tgacaaggca
601 ttaaattatg cggcacaaca ggcggcgagt ctcggtagcc agcttcagtc gcgatctctg
661 aacggcgatt acgcgaaaga taccgctctt ggtatcgctg gtaaccaggc ttcgtcacag
721 ttgcaggcct ggttacaaca ttatggaacg gcagaggtta atctgcagag tggtaataac
781 tttgacggta gttcactgga cttcttatta ccgttctatg attccgaaaa aatgctggca
841 tttggtcagg tcggagcgcg ttacattgac tcccgcttta cggcaaattt aggtgcgggt
901 cagcgttttt tccttcctgc aaacatgttg ggctataacg tcttcattga tcaggatttt
961 tctggtgata atacccgttt aggtattggt ggcgaatact ggcgagacta tttcaaaagt
1021 agcgttaacg gctatttccg catgagcggc tggcatgagt catacaataa gaaagactat
1081 gatgagcgcc cagcaaatgg cttcgatatc cgttttaatg gctatctacc gtcatatccg
1141 gcattaggcg ccaagctgat atatgagcag tattatggtg ataatgttgc tttgtttaat
1201 tctgataagc tgcagtcgaa tcctggtgcg gcgaccgttg gtgtaaacta tactccgatt
1261 cctctggtga cgatggggat cgattaccgt catggtacgg gtaatgaaaa tgatctcctt
1321 tactcaatgc agttccgtta tcagtttgat aaatcgtggt ctcagcaaat tgaaccacag
1381 tatgttaacg agttaagaac attatcaggc agccgttacg atctggttca gcgtaataac
1441 aatattattc tggagtacaa gaagcaggat attctttctc tgaatattcc gcatgatatt
1501 aatggtactg aacacagtac gcagaagatt cagttgatcg ttaagagcaa atacggtctg
1561 gatcgtatcg tctgggatga tagtgcatta cgcagtcagg gcggtcagat tcagcatagc
1621 ggaagccaaa gcgcacaaga ctaccaggct attttgcctg cttatgtgca aggtggcagc
1681 aatatttata aagtgacggc tcgcgcctat gaccgtaatg gcaatagctc taacaatgta
1741 cagcttacta ttaccgttct gtcgaatggt caagttgtcg accaggttgg ggtaacggac
1801 tttacggcgg ataagacttc ggctaaagcg gataacgccg ataccattac ttataccgcg
1861 acggtgaaaa agaatggggt agctcaggct aatgtccctg tttcatttaa tattgtttca
1921 ggaactgcaa ctcttggggc aaatagtgcc aaaacggatg ctaacggtaa ggcaaccgta
1981 acgttgaagt cgagtacgcc aggacaggtc gtcgtgtctg ctaaaaccgc ggagatgact
2041 tcagcactta atgccagtgc ggttatattt tttgatggtg cgccggtgcc gtatccggat
2101 ccgctggaac cggcccagcc ggccatggcg catcaccatc accatcacgc ggccgccgaa
2161 caaaaactca tctcagaaga ggatgcagct gcataaaagc ttgacctgtg aagtgaaaaa
2221 tggcgcacat tgtgcgacat tttttttgtc tgccgtttac cgctactgcg tcacggatcc
2281 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
2341 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
2401 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
2461 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
2521 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
2581 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
2641 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
2701 taacgcgaat tttaacaaaa tattaacgct tacaatttca ggtggcactt ttcggggaaa
2761 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
2821 gtcgagacgt tgggtgaggt tccaactttc accataatga aataagatca ctaccgggcg
2881 tattttttga gttatcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca
2941 ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc
3001 agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa
3061 agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc
3121 tgatgaatgc tcatccggag ttccgtatgg caatgaaaga cggtgagctg gtgatatggg
3181 atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct
3241 ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt
3301 gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct
3361 cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact
3421 tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc
3481 cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta
3541 atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttttt taaggcagtt
3601 attggtgccc ttaaacgcct ggttgctacg cctgaataag tgataataag cggatgaatg
3661 gcagaaattc gaaagcaaat tcgacccggt cgtcggttca gggcagggtc gttaaatagc
3721 cgcttatgtc tattgctggt ttaccggttt attgactacc ggaagcagtg tgaccgtgtg
3781 cttctcaaat gcctgaggcc agtttgctca ggctctcccc gtggaggtaa taattgctcg
3841 aatgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
3901 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
3961 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
4021 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
4081 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
4141 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
4201 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
4261 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
4321 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
4381 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
4441 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
4501 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
4561 catgacccga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg
4621 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
4681 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
4741 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
4801 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
4861 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
4921 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
4981 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
5041 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
5101 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
5161 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
5221 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
5281 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
5341 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
5401 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggacatc tcggtagtgg
5461 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg
5521 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
5581 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
5641 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
5701 aggtttcccg actggaaagc gggcagtgag cggtacccga taaaagcggc ttcctgacag
5761 gaggccgttt tgttttgcag cccacctcaa cgcaattaat gtgagttagc tcactcatta
5821 ggcaccccag gc
//
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