| 质粒名称: | pMAK464 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 140702 |
| 存储实验室: | Takayuki Iwaki |
|
载体骨架基本信息 |
|
| 载体名称: | pMal-T-Avi-His/BirA |
| 载体出品公司: | Clontech |
| 完整质粒大小: | 6460 bp |
| 载体类型: | 细菌表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 未知 |
|
插入基因信息 |
|
| 插入基因名称: | NBαEGFP |
| 克隆方法: | Ligation Independent Cloning |
| 5’测序引物 | caatttcacacaggaaacagccag |
| 3’测序引物 | gtcctactcaggagagcgttc |
|
文献引用方法 |
|
| 材料和方法部分: |
pMAK464 was a gift from Takayuki Iwaki (Addgene plasmid # 140702 ; http://n2t.net/addgene:140702 ; RRID:Addgene_140702)
|
| 参考文献部分: | Nanobody production can be simplified by direct secretion from Escherichia coli. Iwaki T, Hara K, Umemura K. Protein Expr Purif. 2020 Feb 13;170:105607. doi: 10.1016/j.pep.2020.105607. 10.1016/j.pep.2020.105607 PubMed 32062022 |
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 pMAK464 6460 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION NB-alpha-EGFP.
ACCESSION .
VERSION .
KEYWORDS pMAK464
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6460)
AUTHORS Iwaki T, Hara K, Umemura K
TITLE Nanobody production can be simplified by direct secretion from
Escherichia coli.
JOURNAL Protein Expr Purif. 2020 Feb 13;170:105607. doi:
10.1016/j.pep.2020.105607.
PUBMED 32062022
REFERENCE 2 (bases 1 to 6460)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6460
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 318..346
/label=tac promoter
/note="strong E. coli promoter; hybrid between the trp and
lac UV5 promoters"
protein_bind 354..370
/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 359..381
/label=M13/pUC Reverse
/note="In lacZ gene"
CDS 539..877
/codon_start=1
/product="GFP-binding fragment of a single-chain camelid
antibody (Rothbauer et al., 2008)"
/label=GFP nanobody
/translation="VQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKERE
WVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYW
GQGTQVTVS"
CDS 932..949
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 959..982
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
terminator 994..1080
/gene="Escherichia coli rrnB"
/label=rrnB T1 terminator
/note="transcription terminator T1 from the E. coli rrnB
gene"
terminator 1172..1199
/label=rrnB T2 terminator
/note="transcription terminator T2 from the E. coli rrnB
gene"
promoter 1227..1317
/gene="bla"
/label=AmpR promoter
CDS 1318..2178
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(1536..1555)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 2266..2854
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 2755..2774
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 3020..3039
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
protein_bind 3142..3166
/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)."
RBS 3181..3203
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 3210..4172
/codon_start=1
/gene="birA"
/product="E. coli biotin protein ligase"
/label=BirA
/translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
CDS 4211..4228
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(4281..4299)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 4295..4342
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
primer_bind 4472..4489
/label=L4440
/note="L4440 vector, forward primer"
primer_bind 4672..4694
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(4688..4879)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
promoter 5375..5452
/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 5453..75
/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(5472..5491)
/label=LacI-R
/note="LacI, reverse primer"
ORIGIN
1 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
61 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacaatt
121 ctcatgtttg acagcttatc atcgactgca cggtgcacca atgcttctgg cgtcaggcag
181 ccatcggaag ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc
241 aaggcgcact cccgttctgg ataatgtttt ttgcgccgac atcataacgg ttctggcaaa
301 tattctgaaa tgagctgttg acaattaatc atcggctcgt ataatgtgtg gaattgtgag
361 cggataacaa tttcacacag gaaacagcca gtccgtttag gtgttttcac gagcaattga
421 ccaacaagga ccatagatta tgaaaataaa aacaggtgca cgcatcctcg cattatccgc
481 attaacgacg atgatgtttt ccgcctcggc tctcgccaaa atcggatcca tggcgcaggt
541 tcagctggtt gaaagcggtg gtgcactggt tcagcctggt ggtagcctgc gtctgagctg
601 tgcagcaagc ggttttccgg ttaatcgtta tagcatgcgt tggtatcgtc aggcaccggg
661 taaagaacgt gaatgggttg caggtatgag cagtgccggt gatcgtagca gctatgaaga
721 tagcgttaaa ggtcgtttta ccatcagccg tgatgatgca cgtaataccg tttatctgca
781 aatgaatagc ctgaaaccgg aagataccgc agtgtattat tgcaatgtta acgtgggctt
841 tgaatattgg ggtcagggca cccaggttac cgttagcgat atcggactga acgacatctt
901 cgaagctcag aaaatcgaat ggcacggctc ccatcaccat catcaccact aagaattcga
961 ttacaaggat gacgacgata agtaaataag cttcaaataa aacgaaaggc tcagtcgaaa
1021 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
1081 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc
1141 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt
1201 gcgtttctac aaactctttc ggtccgttgt ttatttttct aaatacattc aaatatgtat
1261 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
1321 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
1381 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
1441 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
1501 gaacgtttcc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
1561 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
1621 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
1681 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
1741 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
1801 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
1861 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
1921 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
1981 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
2041 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
2101 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
2161 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgatttc
2221 cttaggactg agcgtcaacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
2281 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
2341 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
2401 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
2461 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
2521 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
2581 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
2641 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
2701 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
2761 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
2821 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
2881 gttcctggcc ttttgctggc cttttgctca catgttccgg taccgcgccc aacagtcccc
2941 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
3001 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
3061 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
3121 attaatacga ctcactagag gggaattgtg agcggataac aattcccctc tagaaataat
3181 tttgtttaac tttaagaagg agatatacca tgaaggataa caccgtgcca ctgaaattga
3241 ttgccctgtt agcgaacggt gaatttcact ctggcgagca gttgggtgaa acgctgggaa
3301 tgagccgggc ggctattaat aaacacattc agacactgcg tgactggggc gttgatgtct
3361 ttaccgttcc gggtaaagga tacagcctgc ctgagcctat ccagttactt aatgctaaac
3421 agatattggg tcagctggat ggcggtagtg tagccgtgct gccagtgatt gactccacga
3481 atcagtacct tcttgatcgt atcggagagc ttaaatcggg cgatgcttgc attgcagaat
3541 accagcaggc tggccgtggt cgccggggtc ggaaatggtt ttcgcctttt ggcgcaaact
3601 tatatttgtc gatgttctgg cgtctggaac aaggcccggc ggcggcgatt ggtttaagtc
3661 tggttatcgg tatcgtgatg gcggaagtat tacgcaagct gggtgcagat aaagttcgtg
3721 ttaaatggcc taatgacctc tatctgcagg atcgcaagct ggcaggcatt ctggtggagc
3781 tgactggcaa aactggcgat gcggcgcaaa tagtcattgg agccgggatc aacatggcaa
3841 tgcgccgtgt tgaagagagt gtcgttaatc aggggtggat cacgctgcag gaagcgggga
3901 tcaatctcga tcgtaatacg ttggcggcca tgctaatacg tgaattacgt gctgcgttgg
3961 aactcttcga acaagaagga ttggcacctt atctgtcgcg ctgggaaaag ctggataatt
4021 ttattaatcg cccagtgaaa cttatcattg gtgataaaga aatatttggc atttcacgcg
4081 gaatagacaa acagggggct ttattacttg agcaggatgg aataataaaa ccctggatgg
4141 gcggtgaaat atccctgcgt agtgcagaaa aaggtaagct ttgatgagga ctagtgcggc
4201 cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga
4261 agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
4321 acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatgct agcacatgtt
4381 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
4441 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
4501 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatataagg
4561 tgcactgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
4621 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
4681 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
4741 catcagcgtg gtcgtgcagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
4801 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
4861 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
4921 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
4981 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
5041 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
5101 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
5161 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
5221 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
5281 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
5341 cccgtggcca ggacccaacg ctgcccgaaa ttccgacacc atcgaatggt gcaaaacctt
5401 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc
5461 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
5521 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
5581 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
5641 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
5701 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
5761 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
5821 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
5881 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
5941 tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
6001 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
6061 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
6121 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
6181 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
6241 cgttggtgcg gatatttcgg tagtgggata cgacgatacc gaagacagct catgttatat
6301 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
6361 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact
6421 ggtgaaaaga aaaaccaccc tggcgcccaa tacacaaacc
//
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