| 质粒名称: | pASKt15C+SrtA |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 64983 |
| 存储实验室: | Teruyuki Nagamune |
|
载体骨架基本信息 |
|
| 载体名称: | pASK-IBA3 plus |
| 载体出品公司: | IBA |
| 载体抗性: | 氯霉素 |
| 空载体大小: | 3211 bp |
| 完整质粒大小: | 3772 bp |
| 载体修饰: | 氨苄西林耐药基因被氯霉素耐药基因取代。用p15A复制子替换pBR322复制子。 |
| 载体类型: | 细菌表达 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | sortase A,SrtA |
| 物种来源: | Staphylococcus aureus ATCC 10832 |
| 插入大小: | 566 bp |
| 基因突变: | deleted amino acids 1-59 |
| 启动子 | tetracycline promoter/operator |
| 标签/融合蛋白: |
His6 tag（插入件上的N端子）
S tag（插入件上的N端子）
|
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | NcoI(未破坏) |
| 3' 克隆位点: | PmeI(未破坏) |
| 5' 测序引物 | GAGTTATTTTACCACTCCCT |
| 3' 测序引物 | CGCAGTAGCGGTAAACG |
|
文献引用方法 |
|
| 材料和方法部分: | pASKt15C+SrtA was a gift from Teruyuki Nagamune (Addgene plasmid # 64983 ; http://n2t.net/addgene:64983 ; RRID:Addgene_64983) |
| 参考文献部分: | Ca -independent sortase-A exhibits high selective protein ligation activity in the cytoplasm of E. coli. Hirakawa H, Ishikawa S, Nagamune T. Biotechnol J. 2015 Apr 10. doi: 10.1002/biot.201500012. 10.1002/biot.201500012 PubMed 25864513 |
买家导航
pASKt15CSrtA载体基本信息
pASKt15CSrtA载体图谱质粒图谱和多克隆位点信息
pASKt15CSrtA载体简介
pASKt15CSrtA载体序列
LOCUS pASKt15C+SrtA 3772 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Expresses the wild type of Staphylococcus aureus sortase A in E.
coli.
ACCESSION .
VERSION .
KEYWORDS pASKt15C+SrtA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3772)
AUTHORS Hirakawa H, Ishikawa S, Nagamune T
TITLE Ca -independent sortase-A exhibits high selective protein ligation
activity in the cytoplasm of E. coli.
JOURNAL Biotechnol J. 2015 Apr 10. doi: 10.1002/biot.201500012.
PUBMED 25864513
REFERENCE 2 (bases 1 to 3772)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..3772
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 36..91
/gene="tetR"
/label=tetR/tetA promoters
/note="overlapping promoters for bacterial tetR and tetA"
protein_bind 42..60
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O1 for the tetR and tetA genes"
protein_bind 72..90
/gene="tetO"
/label=tet operator
/bound_moiety="tetracycline repressor TetR"
/note="bacterial operator O2 for the tetR and tetA genes"
RBS 130..152
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 165..182
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 192..209
/codon_start=1
/product="thrombin recognition and cleavage site"
/label=thrombin site
/translation="LVPRGS"
CDS 216..260
/codon_start=1
/product="affinity and epitope tag derived from pancreatic
ribonuclease A"
/label=S-Tag
/translation="KETAAAKFERQHMDS"
rep_origin 808..1236
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(895..914)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 1105..1126
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 1265..1367
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
CDS 1368..2027
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
primer_bind complement(1434..1453)
/label=CAT-R
/note="Chloramphenicol resistance gene, reverse primer"
CDS 2037..2663
/codon_start=1
/gene="tetR"
/product="tetracycline repressor TetR"
/label=TetR
/note="TetR binds to the tetracycline operator tetO to
inhibit transcription. This inhibition can be relieved by
adding tetracycline or doxycycline."
/translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
primer_bind complement(2113..2132)
/label=Tet-R
/note="Tetracycline resistance gene, reverse primer"
primer_bind complement(2934..2951)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(3068..3613)
/direction=LEFT
/label=p15A ori
/note="Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin.
"
primer_bind complement(3148..3167)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 ccatcgaatg gccagatgat taattcctaa tttttgttga cactctatca ttgatagagt
61 tattttacca ctccctatca gtgatagaga aaagtgaaat gaatagttcg acaaaaatct
121 agaaataatt ttgtttaact ttaagaagga gatataccat gggccaccat catcatcatc
181 attcttctgg tctggtgcca cgcggttctg gtatgaaaga aaccgctgct gctaaattcg
241 aacgccagca catggacagc ccagatctgg gtacccaagc taaacctcaa attccgaaag
301 ataaatcgaa agtggcaggc tatattgaaa ttccagatgc tgatattaaa gaaccagtat
361 atccaggacc agcaacacct gaacaattaa atagaggtgt aagctttgca gaagaaaacg
421 aatcactaga tgatcaaaat atttcaattg caggacacac tttcattgac cgtccgaact
481 atcaatttac aaatcttaaa gcagccaaaa aaggtagtat ggtgtacttt aaagttggta
541 atgaaacacg taagtataaa atgacaagta taagagatgt taagcctaca gatgtagaag
601 ttctagatga acaaaaaggt aaagataaac aattaacatt aattacttgt gatgattaca
661 atgaaaagac aggcgtttgg gaaaaacgta aaatctttgt agctacagaa gtcaaataag
721 tttaaacgac ctgtgaagtg aaaaatggcg cacattgtgc gacatttttt ttgtctgccg
781 tttaccgcta ctgcgtcacg gatctccacg cgccctgtag cggcgcatta agcgcggcgg
841 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
901 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
961 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
1021 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
1081 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
1141 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
1201 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatttgg cgaaaatgag
1261 acgttgatcg gcacgtaaga ggttccaact ttcaccataa tgaaataaga tcactaccgg
1321 gcgtattttt tgagttatcg agattttcag gagctaagga agctaaaatg gagaaaaaaa
1381 tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat
1441 ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt
1501 taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc
1561 gcctgatgaa tgctcatccg gaatttcgta tggcaatgaa agacggtgag ctggtgatat
1621 gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc
1681 tctggagtga ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg
1741 cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg
1801 tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca
1861 acttcttcgc ccccgttttc actatgggca aatattatac gcaaggcgac aaggtgctga
1921 tgccgctggc gattcaggtt catcatgccg tctgtgatgg cttccatgtc ggcagaatgc
1981 ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg ggcgtaatag gaattaatga
2041 tgtctcgttt agataaaagt aaagtgatta acagcgcatt agagctgctt aatgaggtcg
2101 gaatcgaagg tttaacaacc cgtaaactcg cccagaagct aggtgtagag cagcctacat
2161 tgtattggca tgtaaaaaat aagcgggctt tgctcgacgc cttagccatt gagatgttag
2221 ataggcacca tactcacttt tgccctttag aaggggaaag ctggcaagat tttttacgta
2281 ataacgctaa aagttttaga tgtgctttac taagtcatcg cgatggagca aaagtacatt
2341 taggtacacg gcctacagaa aaacagtatg aaactctcga aaatcaatta gcctttttat
2401 gccaacaagg tttttcacta gagaatgcat tatatgcact cagcgcagtg gggcatttta
2461 ctttaggttg cgtattggaa gatcaagagc atcaagtcgc taaagaagaa agggaaacac
2521 ctactactga tagtatgccg ccattattac gacaagctat cgaattattt gatcaccaag
2581 gtgcagagcc agccttctta ttcggccttg aattgatcat atgcggatta gaaaaacaac
2641 ttaaatgtga aagtgggtct taaaagcagc ataacctttt tccgtgatgg taacttcact
2701 agtttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
2761 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gcgctagcgg agtgtatact
2821 ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa
2881 aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc
2941 actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc
3001 ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa
3061 agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc
3121 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc
3181 tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc
3241 gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac
3301 tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt
3361 gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt
3421 agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg
3481 tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt
3541 cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc
3601 aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga tttcagtgca
3661 atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt tgtaattctg
3721 cctttttacg gttcctggcc ttttgctggc cttttgctca catgacccga ca
//
