| 质粒名称: | pCA24N-p50-ligase |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 87742 |
| 存储实验室: | Wayne Patrick |
|
载体骨架基本信息 |
|
| 载体名称: | pCA24N |
| 载体出品公司: | Kitagawa et al. (PMID:16769691) |
| 载体抗性: | 氯霉素 |
| 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: | -- |
| 高拷贝/低拷贝: | -- |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | T4 DNA ligase |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | T5-lac |
| 标签/融合蛋白: |
6xHis（主干上的N终端）
humanp50/NF-kB（氨基酸40-366）（插入端N端）
|
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | SfiI(未破坏) |
| 3' 克隆位点: | SfiI(未破坏) |
| 5' 测序引物 | pCA24N.for (GATAACAATTTCACACAGAATTCATTAAAGAG) |
| 3' 测序引物 | pCA24N.rev2 — 5'-CAAATCCAGATGGAGTTCTGAGG-3' |
|
文献引用方法 |
|
| 材料和方法部分: | pCA24N-p50-ligase was a gift from Wayne Patrick (Addgene plasmid # 87742 ; http://n2t.net/addgene:87742 ; RRID:Addgene_87742) |
| 参考文献部分: | Engineered DNA ligases with improved activities in vitro. Wilson RH, Morton SK, Deiderick H, Gerth ML, Paul HA, Gerber I, Patel A, Ellington AD, Hunicke-Smith SP, Patrick WM. Protein Eng Des Sel. 2013 Jul;26(7):471-8. doi: 10.1093/protein/gzt024. Epub 2013 Jun 10. 10.1093/protein/gzt024 PubMed 23754529 |
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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 pCA24N-p50-ligase 7006 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION IPTG-inducible expression of p50-ligase fusion for protein
purification.
ACCESSION .
VERSION .
KEYWORDS pCA24N-p50-ligase
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7006)
AUTHORS Wilson RH, Morton SK, Deiderick H, Gerth ML, Paul HA, Gerber I,
Patel A, Ellington AD, Hunicke-Smith SP, Patrick WM
TITLE Engineered DNA ligases with improved activities in vitro.
JOURNAL Protein Eng Des Sel. 2013 Jul;26(7):471-8. doi:
10.1093/protein/gzt024. Epub 2013 Jun 10.
PUBMED 23754529
REFERENCE 2 (bases 1 to 7006)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7006
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 80..124
/label=T5 promoter
/note="bacteriophage T5 promoter for E. coli RNA
polymerase, with embedded lac operator"
protein_bind 100..116
/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)."
protein_bind 132..148
/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 167..178
/note="strong bacterial ribosome binding site (Elowitz and
Leibler, 2000)"
CDS 197..214
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
terminator 2772..2866
/label=lambda t0 terminator
/note="transcription terminator from phage lambda"
primer_bind complement(2936..2958)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
promoter 3422..3524
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
CDS 3525..4184
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
primer_bind complement(3591..3610)
/label=CAT-R
/note="Chloramphenicol resistance gene, reverse primer"
rep_origin 4735..5323
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5224..5243
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 5477..5494
/label=L4440
/note="L4440 vector, forward primer"
promoter 5567..5644
/gene="lacI"
/label=lacI promoter
CDS 5645..6727
/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(5664..5683)
/label=LacI-R
/note="LacI, reverse primer"
promoter 6776..6806
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6814..6830
/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 6819..6841
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 6838..6854
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 6838..6854
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(6870..6887)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(6870..6886)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(6879..6901)
/label=M13/pUC Forward
/note="In lacZ gene"
ORIGIN
1 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
61 tcgtcttcac ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat
121 aatagattca attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt
181 aactatgaga ggatctcacc atcaccatca ccatacggat ccggccctga gggccgcaga
241 tggcccatac cttcaaatat tagagcaacc taaacagaga ggatttcgtt tccgttatgt
301 atgtgaaggc ccatcccatg gtggattacc tggtgcctct agtgaaaaga acaagaagtc
361 ttaccctcag gtcaaaatct gcaactatgt gggaccagca aaggttattg ttcagttggt
421 cacaaatgga aaaaatatcc acctgcatgc ccacagcctg gtgggaaaac actgtgagga
481 tgggatctgc actgtaactg ctggacccaa ggacatggtg gtcggcttcg caaacctggg
541 tatacttcat gtgacaaaga aaaaagtatt tgaaacactg gaagcacgaa tgacagaggc
601 gtgtataagg ggctataatc ctggactctt ggtgcaccct gaccttgcct atttgcaagc
661 agaaggtgga ggggaccggc agctgggaga tcgggaaaaa gagctaatcc gccaagcagc
721 tctgcagcag accaaggaga tggacctcag cgtggtgcgg ctcatgttta cagcttttct
781 tccggatagc actggcagct tcacaaggcg cctggaaccc gtggtatcag acgccatcta
841 tgacagtaaa gcccccaatg catccaactt gaaaattgta agaatggaca ggacagctgg
901 atgtgtgact ggaggggagg aaatttatct tctttgtgac aaagttcaga aagatgacat
961 ccagattcga ttttatgaag aggaagaaaa tggtggagtc tgggaaggat ttggagattt
1021 ttcccccaca gatgttcata gacaatttgc cattgtcttc aaaactccaa agtataaaga
1081 tattaatatt acaaaaccag cctctgtgtt tgtccagctt cggaggaaat ctgacttgga
1141 aaccagtgaa ccaaaacctt tcctctacta tcctgaaatc aaagataaag aagaagtgca
1201 gaggaaacgt cagaagggga gctcgggcac tagtggcgga ggctccggcg gtggcattct
1261 taaaattctg aacgaaatag catctattgg ttcaactaaa cagaagcaag caattcttga
1321 aaagaataaa gataatgaat tgcttaaacg agtatatcgt ctgacttatt ctcgtgggtt
1381 acagtattat atcaagaaat ggcctaaacc tggtattgct acccagagtt ttggaatgtt
1441 gactcttacc gatatgcttg acttcattga attcacatta gctactcgga aattgactgg
1501 aaatgcagca attgaggaat taactggata tatcaccgat ggtaaaaaag atgatgttga
1561 agttttgcgt cgagtgatga tgcgagacct tgaatgtggt gcttcagtat ctattgcaaa
1621 caaagtttgg ccaggtttaa ttcctgaaca acctcaaatg ctcgcaagtt cttatgatga
1681 aaaaggcatt aataagaata tcaaatttcc agcctttgct cagttaaaag ctgatggagc
1741 tcggtgtttt gctgaagtta gaggtgatga attagatgat gttcgtcttt tatcacgagc
1801 tggtaatgaa tatctaggat tagatcttct taaggaagag ttaattaaaa tgaccgctga
1861 agcccgccag attcatccag aaggtgtgtt gattgatggc gaattggtat accatgagca
1921 agttaaaaag gagccagaag gcctagattt tctttttgat gcttatcctg aaaacagtaa
1981 agctaaagaa ttcgccgaag ttgctgaatc acgtactgct tctaatggaa tcgccaataa
2041 atctttaaag ggaaccattt ctgaaaaaga agcacaatgc atgaagtttc aggtctggga
2101 ttatgtcccg ttggtagaaa tatacagtct tcctgcattt cgtttgaaat atgatgtacg
2161 tttttctaaa ctagaacaaa tgacatctgg atatgataaa gtaattttaa ttgaaaacca
2221 ggtagtaaat aacctagatg aagctaaggt aatttataaa aagtatattg accaaggtct
2281 tgaaggtatt attctcaaaa atatcgatgg attatgggaa aatgctcgtt caaaaaatct
2341 ttataaattt aaagaagtaa ttgatgttga tttaaaaatt gtaggaattt atcctcaccg
2401 taaagaccct actaaagcgg gtggatttat tcttgagtca gagtgtggaa aaattaaggt
2461 aaatgctggt tcaggcttaa aagataaagc cggtgtaaaa tcgcatgaac ttgaccgtac
2521 tcgcattatg gaaaaccaaa attattatat tggaaaaatt ctagagtgcg aatgcaacgg
2581 ttggttaaaa tctgatggcc gcactgatta cgttaaatta tttcttccga ttgcgattcg
2641 tttacgtgaa gataaaacta aagctaatac attcgaagat gtatttggtg attttcatga
2701 ggtaactggt ctataaggcc tatgcggccg ctaagggtcg acctgcagcc aagcttaatt
2761 agctgagctt ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt
2821 gttcagaacg ctcggttgcc gccgggcgtt ttttattggt gagaatccaa gctaggggaa
2881 ttcatcgtga ctgactgacg atctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
2941 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
3001 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
3061 cacgtagcga tagcggagtg tataattctt gaagacgaaa gggcctcgtg atacgcctat
3121 ttttataggt taatgtcatg ataataatag tttcttagaa tcctgatgtc cggcggtgct
3181 tttgccgtta cgcaccaccc cgtcagtagc tgaacaggag ggacagctga tagaaacaga
3241 agccactgga gcacctcaaa aacaccatca tacactaaat cagtaagttg gcagcatcac
3301 ccgacgcact ttgcgccgaa taaatacctg tgacggaaga tcacttcgca gaataaataa
3361 atcctggtgt ccctgttgat accgggaagc cctgggccaa cttttggcga aaatgagacg
3421 ttgatcggca cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg
3481 tattttttga gttatcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca
3541 ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc
3601 agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa
3661 agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc
3721 tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg
3781 atagtgttca cccttgttat accgttttcc atgagcaaac tgaaacgttt tcatcgctct
3841 ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt
3901 gttacggtga gaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct
3961 cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact
4021 tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc
4081 cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta
4141 atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttttt taaggcagtt
4201 attggtgccc ttaaacgcct ggttgctacg cctgaataag tgataataag cggatgaatg
4261 gcagaaattc gaaagcaaat tcgacccggt cgtcggttca gggcagggtc gttaaatagc
4321 cgcttatgtc tattgctggt ttaccggttt attgactacc ggaagcagtg tgaccgtgtg
4381 cttctcaaat gcctgaggcc agtttgctca ggctctcccc gtggaggtaa taattgacga
4441 tatgatcatt tattctgcct cccagagcct gataaaaacg gttaccggat cgagtcaggc
4501 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
4561 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
4621 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
4681 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
4741 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
4801 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
4861 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
4921 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
4981 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
5041 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
5101 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
5161 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
5221 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
5281 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
5341 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
5401 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
5461 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
5521 ttttctcctt acgcatctgt gcggtatttc acaccgcata aattccgaca ccatcgaatg
5581 gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
5641 gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
5701 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
5761 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
5821 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
5881 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
5941 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
6001 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
6061 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
6121 tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca
6181 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
6241 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
6301 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
6361 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
6421 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag
6481 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
6541 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
6601 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
6661 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
6721 gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccc caggctttac
6781 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
6841 gaaacagcta tgaccatgat tacggattca ctggccgtcg ttttacaacg tcgtgactgg
6901 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
6961 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgc
//
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