| 质粒名称: | pEHlyA5 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 168298 |
| 存储实验室: | Luis Ángel Fernández |
|
载体骨架基本信息 |
|
| 载体名称: | pUC18 |
| 载体出品公司: | Joachim Messing |
| 空载体大小: | 2712 bp |
| 完整质粒大小: | 3991 bp |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 载体抗性: | 氨苄霉素100 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: | N‑terminal His tag, VHH, HA and E‑tags, C‑HlyA |
| 物种来源: | Synthetic |
| 插入大小: | 1279 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | LacI-Plac |
| 标签/融合蛋白: |
His-tag （插件上的N端子）
VHH Amy（插接件上的N端子）
HA-tag（插接件上的N端子）
Prescission site（插页上的N端子）
E-tag（插件上的N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: | pEHlyA5 was a gift from Luis Ángel Fernández (Addgene plasmid # 168298 ; http://n2t.net/addgene:168298 ; RRID:Addgene_168298) |
| 参考文献部分: | Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system. Ruano-Gallego D, Fraile S, Gutierrez C, Fernandez LA. Microb Cell Fact. 2019 Mar 11;18(1):47. doi: 10.1186/s12934-019-1094-0. 10.1186/s12934-019-1094-0 PubMed 30857538 |
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 pEHlyA5 3990 bp ds-DNA circular SYN 09-JUN-2021
DEFINITION Secretion of nanobodies and other polypeptides fused to C-terminal
secretion signal of HlyA.
ACCESSION .
VERSION .
KEYWORDS pEHlyA5
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3990)
AUTHORS Ruano-Gallego D, Fraile S, Gutierrez C, Fernandez LA
TITLE Screening and purification of nanobodies from E. coli culture
supernatants using the hemolysin secretion system.
JOURNAL Microb Cell Fact. 2019 Mar 11;18(1):47. doi:
10.1186/s12934-019-1094-0.
PUBMED 30857538
REFERENCE 2 (bases 1 to 3990)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Jun 9, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..3990
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 123..142
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 376..393
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 510..531
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 546..576
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 584..600
/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 589..611
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 608..624
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 608..624
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 666..685
/label=T7
/note="T7 promoter, forward primer"
promoter 666..684
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
RBS 716..738
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 752..769
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 1184..1210
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 1211..1234
/codon_start=1
/product="recognition and cleavage site for human
rhinovirus 3C and PreScission proteases"
/label=HRV 3C site
/translation="LEVLFQGP"
primer_bind complement(1998..2015)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(1998..2014)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2007..2029)
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind complement(2223..2242)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 2342..2364
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(2402..2420)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 2488..2592
/gene="bla"
/label=AmpR promoter
CDS 2593..3453
/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(2811..2830)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 3624..222
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
ORIGIN
1 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
61 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
121 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
181 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
241 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
301 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
361 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
421 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
481 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
541 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
601 tttcacacag gaaacagcta tgacatgatt acgaatttag atctgaattc gggcccttcg
661 aaaattaata cgactcacta tagggagacc acaacggttt ccctctagaa ataattttgt
721 ttaactttaa gaaggagata tattcatggc tcatcaccat caccatcacg cggcccagcc
781 ggccatggct caggtgcagc tggtggagtc ttggggaggc tcggtgcagg ctggggggtc
841 tctgagactc tcctgcacag cccctggatt cacctccaat agctgccgca tggactggta
901 ccgccaggct gcagggaagc agcgcgagtg ggtctcatct attagtactg atggtcgcac
961 aagctatgca gactccgtga agggccgatt caccatctcc aaagacaaag ccaaggacac
1021 ggtgtatctg caaatgaaca gcctgaaacc tgaggacacg gccatctatt actgtgccgt
1081 gaggacgaat gggtatcgtc cgcaatctca cgaatttcgc tactggggcc cggggaccca
1141 ggtcaccgtc tcctcagcgg ccgcatcggg ggccgcgtcg acgtacccat acgatgttcc
1201 agattacgct ctggaagttc tgttccaggg gcccgcttcg acgcccgggg gtgcgccggt
1261 gccgtatccg gatccgctgg aaccggccgg ggaaaattct cttgctaaaa atgtattatc
1321 cggtggaaaa ggtaatgaca agttgtacgg cagtgaggga gcagacctgc ttgatggcgg
1381 agaagggaat gatcttctga aaggtggata tggtaatgat atttatcgtt atctttcagg
1441 atatggccat catattattg acgatgaagg ggggaaagac gataaactca gtttagctga
1501 tatagatttc cgggacgttg cctttaagcg agaagggaat gacctcatta tgtataaagc
1561 tgaaggtaat gttctttcta ttggccacaa aaatggtatt acatttaaaa actggtttga
1621 aaaagagtca gatgatctct ctaatcatca gatagagcag atttttgata aagacggcag
1681 ggtaatcaca ccagattctc ttaaaaaagc atttgaatat cagcagagta ataacaaggt
1741 aagttatgtg tatggacatg atgcatcaac ttatgggagc caggacaatc ttaatccatt
1801 aattaatgaa atcagcaaaa tcatttcagc tgcaggtaac ttcgatgtta aggaggaaag
1861 atctgccgct tctttattgc agttgtccgg taatgccagt gatttttcat atggacggaa
1921 ctcaataact ttgacagcat cagcataata tattaattta aatgatagca atcttactgg
1981 gctgtgccaa gcttggcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
2041 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
2101 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg
2161 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc
2221 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
2281 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
2341 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
2401 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
2461 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
2521 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
2581 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
2641 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
2701 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
2761 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
2821 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
2881 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
2941 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
3001 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
3061 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
3121 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
3181 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
3241 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
3301 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
3361 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
3421 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
3481 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
3541 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
3601 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
3661 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
3721 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
3781 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
3841 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
3901 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
3961 cagcccagct tggagcgaac gacctacacc
//
