| 质粒名称: | pCA24N-sso7d-ligase |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 87745 |
| 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) |
| 空载体大小: | -- |
| 完整质粒大小: | 6202 bp |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 载体抗性: | 氯霉素，25μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | -- |
|
插入基因信息 |
|
| 插入基因名称: | T4 DNA连接酶 |
| 物种来源: | -- |
| 插入大小: | 951 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | T5-lac |
| 标签/融合蛋白: | 6xHis（主干上的N端子）密码子优化的sso7d（苏富龙）（delAA1-4）（插入物上的N末端） |
| 克隆方法: | -- |
| 5’克隆位点: | -- |
| 3’克隆位点: | -- |
| 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-sso7d-ligase was a gift from Wayne Patrick (Addgene plasmid # 87745 ; http://n2t.net/addgene:87745 ; RRID:Addgene_87745) |
| 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 |
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 pCA24N-sso7d-ligase 6202 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION IPTG-inducible expression of sso7d-ligase fusion for protein
purification.
ACCESSION .
VERSION .
KEYWORDS pCA24N-sso7d-ligase
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6202)
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 6202)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6202
/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 1968..2062
/label=lambda t0 terminator
/note="transcription terminator from phage lambda"
primer_bind complement(2132..2154)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
promoter 2618..2720
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
CDS 2721..3380
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
primer_bind complement(2787..2806)
/label=CAT-R
/note="Chloramphenicol resistance gene, reverse primer"
rep_origin 3931..4519
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4420..4439
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 4673..4690
/label=L4440
/note="L4440 vector, forward primer"
promoter 4763..4840
/gene="lacI"
/label=lacI promoter
CDS 4841..5923
/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(4860..4879)
/label=LacI-R
/note="LacI, reverse primer"
promoter 5972..6002
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6010..6026
/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 6015..6037
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 6034..6050
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 6034..6050
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(6066..6083)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(6066..6082)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(6075..6097)
/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 gggcgactgt
241 taaattcaaa tacaaaggtg aggagaaaga agttgacatt tccaagatca aaaaggtatg
301 gcgcgtgggc aaaatgatct ctttcactta cgacgaaggc ggcggtaaaa ccggtcgtgg
361 cgcggtgagc gaaaaggacg cgccaaaaga actgctgcag atgctggaaa aacagaagaa
421 aggcactagt ggcggaggct ccggcggtgg cattcttaaa attctgaacg aaatagcatc
481 tattggttca actaaacaga agcaagcaat tcttgaaaag aataaagata atgaattgct
541 taaacgagta tattgtctga cttattctcg tgggttacag tattatatca agaaatggcc
601 taaacctggt attgctaccc agagttttgg aatgttgact cttaccgata tgcttgactt
661 cattgaattc acattagcta ctcggaaatt gactggaaat gcagcaattg aggaattaac
721 tggatatatc accgatggta aaaaagatga tgttgaagtt ttgcgtcgag tgatgatgcg
781 agaccttgaa tgtggtgctt cagtatctat tgcaaacaaa gtttggccag gtttaattcc
841 tgaacaacct caaatgctcg caagttctta tgatgaaaaa ggcattaata agaatatcaa
901 atttccagcc tttgctcagt taaaagctga tggagctcgg tgttttgctg aagttagagg
961 tgatgaatta gatgatgttc gtcttttatc acgagctggt aatgaatatc taggattaga
1021 tcttcttaag gaagagttaa ttaaaatgac cgctgaagcc cgccagattc atccagaagg
1081 tgtgttgatt gatggcgaat tggtatacca tgagcaagtt aaaaaggagc cagaaggcct
1141 agattttctt tttgatgctt atcctgaaaa cagtaaagct aaagaattcg ccgaagttgc
1201 tgaatcacgt actgcttcta atggaatcgc caataaatct ttaaagggaa ccatttctga
1261 aaaagaagca caatgcatga agtttcaggt ctgggattat gtcccgttgg tagaaatata
1321 cagtcttcct gcatttcgtt tgaaatatga tgtacgtttt tctaaactag aacaaatgac
1381 atctggatat gataaagtaa ttttaattga aaaccaggta gtaaataacc tagatgaagc
1441 taaggtaatt tataaaaagt atattgacca aggtcttgaa ggtattattc tcaaaaatat
1501 cgatggatta tgggaaaatg ctcgttcaaa aaatctttat aaatttaaag aagtaattga
1561 tgttgattta aaaattgtag gaatttatcc tcaccgtaaa gaccctacta aagcgggtgg
1621 atttattctt gagtcagagt gtggaaaaat taaggtaaat gctggttcag gcttaaaaga
1681 taaagccggt gtaaaatcgc atgaacttga ccgtactcgc attatggaaa accaaaatta
1741 ttatattgga aaaattctag agtgcgaatg caacggttgg ttaaaatctg atggccgcac
1801 tgattacgtt aaattatttc ttccgattgc gattcgttta cgtgaagata aaactaaagc
1861 taatacattc gaagatgtat ttggtgattt tcatgaggta actggtctat aaggcctatg
1921 cggccgctaa gggtcgacct gcagccaagc ttaattagct gagcttggac tcctgttgat
1981 agatccagta atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg
2041 ggcgtttttt attggtgaga atccaagcta ggggaattca tcgtgactga ctgacgatct
2101 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
2161 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
2221 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata
2281 attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
2341 taatagtttc ttagaatcct gatgtccggc ggtgcttttg ccgttacgca ccaccccgtc
2401 agtagctgaa caggagggac agctgataga aacagaagcc actggagcac ctcaaaaaca
2461 ccatcataca ctaaatcagt aagttggcag catcacccga cgcactttgc gccgaataaa
2521 tacctgtgac ggaagatcac ttcgcagaat aaataaatcc tggtgtccct gttgataccg
2581 ggaagccctg ggccaacttt tggcgaaaat gagacgttga tcggcacgta agaggttcca
2641 actttcacca taatgaaata agatcactac cgggcgtatt ttttgagtta tcgagatttt
2701 caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat
2761 cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata
2821 accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca
2881 agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattcc
2941 gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct tgttataccg
3001 ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc
3061 ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgagaac ctggcctatt
3121 tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca
3181 ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg
3241 gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg
3301 ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg
3361 agtggcaggg cggggcgtaa tttttttaag gcagttattg gtgcccttaa acgcctggtt
3421 gctacgcctg aataagtgat aataagcgga tgaatggcag aaattcgaaa gcaaattcga
3481 cccggtcgtc ggttcagggc agggtcgtta aatagccgct tatgtctatt gctggtttac
3541 cggtttattg actaccggaa gcagtgtgac cgtgtgcttc tcaaatgcct gaggccagtt
3601 tgctcaggct ctccccgtgg aggtaataat tgacgatatg atcatttatt ctgcctccca
3661 gagcctgata aaaacggtta ccggatcgag tcaggcaact atggatgaac gaaatagaca
3721 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
3781 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
3841 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
3901 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
3961 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
4021 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
4081 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
4141 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
4201 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
4261 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
4321 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
4381 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
4441 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
4501 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
4561 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
4621 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
4681 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
4741 tatttcacac cgcataaatt ccgacaccat cgaatggcgc aaaacctttc gcggtatggc
4801 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
4861 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
4921 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
4981 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
5041 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
5101 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
5161 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
5221 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
5281 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
5341 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
5401 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
5461 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
5521 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
5581 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
5641 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
5701 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
5761 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
5821 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
5881 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
5941 taagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
6001 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
6061 gattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
6121 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
6181 gatcgccctt cccaacagtt gc
//
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