| 质粒名称: | pQE80L TriCatcher-RGDSP |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 12631 |
| 存储实验室: | Mark Howarth |
|
载体骨架基本信息 |
|
| 载体名称: | pQE80L |
| 载体出品公司: | Clontech |
| 空载体大小: | 4714bp |
| 完整质粒大小: | 6539bp |
| 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: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | TriCatcher-RGDSP |
| 物种来源: | Synthetic |
| 插入大小: | 1836bp |
| 基因突变: | central RGDSP integrin-binding sequence |
| GenBank编号 | JN247409 |
| 启动子: | T5 |
| 标签/融合蛋白: |
His6 (N terminal on insert)
TEV cleavage site (N terminal on insert)
TEV cleavage site
|
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | CATAAAAAATTTATTTGCTTTGTGAGCGG |
| 3’测序引物 | CGAGCGTTCTGAACAAATCC |
|
文献引用方法 |
|
| 材料和方法部分: | pQE80L TriCatcher-RGDSP was a gift from Mark Howarth (Addgene plasmid # 112631 ; http://n2t.net/addgene:112631 ; RRID:Addgene_112631) |
| 参考文献部分: | Assembling and decorating hyaluronan hydrogels with twin protein superglues to mimic cell-cell interactions. Wieduwild R, Howarth M. Biomaterials. 2018 Oct;180:253-264. doi: 10.1016/j.biomaterials.2018.07.020. Epub 2018 Jul 17. 10.1016/j.biomaterials.2018.07.020 PubMed 30053659 |
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LOCUS pQE80L TriCatcher-RGDSP 6539 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Two SpyCatchers linked by an elastin-like polypeptide with an RGDSP integrin-binding site and a C-terminal SnoopCatcher. ACCESSION . VERSION . KEYWORDS pQE80L TriCatcher-RGDSP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6539) AUTHORS Wieduwild R, Howarth M TITLE Assembling and decorating hyaluronan hydrogels with twin protein superglues to mimic cell-cell interactions JOURNAL Biomaterials REFERENCE 2 (bases 1 to 6539) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.biofeng.com FEATURES Location/Qualifiers source 1..6539 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 40..58 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 82..126 /label=T5 promoter /note="bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator" protein_bind 102..118 /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 134..150 /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 169..180 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 202..219 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 238..258 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 1198..1218 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" terminator 2068..2162 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS 2206..2865 /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind complement(2272..2291) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" terminator 2930..3016 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" CDS complement(3109..4191) /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 4153..4172 /label=LacI-R /note="LacI, reverse primer" promoter complement(4192..4269) /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." primer_bind complement(4353..4375) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 4461..4601 /label=bom /note="basis of mobility region from pBR322" primer_bind 4516..4535 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(4616..4633) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4787..5375) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4867..4886) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5546..6406) /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 6169..6188 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6407..6511) /gene="bla" /label=AmpR promoter ORIGIN 1 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 61 tttcgtcttc accttgagaa atcataaaaa atttatttgc tttgtgagcg gataacaatt 121 ataatagatt caattgtgag cggataacaa tttcacacag aattcattaa agaggagaaa 181 ttaactatga aaggcagcag ccatcatcat catcatcacg tcgacatccc aacgaccgaa 241 aacctgtatt ttcagggcgc catggttgat accttatcag gtttatcaag tgagcaaggt 301 cagtccggtg atatgacaat tgaagaagat agtgctaccc atattaaatt ctcaaaacgt 361 gatgaggacg gcaaagagtt agctggtgca actatggagt tgcgtgattc atctggtaaa 421 actattagta catggatttc agatggacaa gtgaaagatt tctacctgta tccaggaaaa 481 tatacatttg tcgaaaccgc agcaccagac ggttatgagg tagcaactgc tattaccttt 541 acagttaatg agcaaggtca ggttactgta aatggcaaag caactaaagg tgacgctcat 601 attgacggta gcggtggctc tggcggtcag ctcgacggcc acggcgtggg tgttccgggc 661 gtcggtgtgc cgggtgtggg tgtgccgggc gaaggtgtgc cgggcgtcgg tgtgccgggt 721 gttggtgttc cgggcgttgg tgtgccgggc gttggcgtgc cgggcgaggg tgtgccgggc 781 gttggtgttc cgggcgtggg tgtgccgggc gtgggcgtgc cgggcgtcgg tgttccgggc 841 gagggtgtgc cgggcgtagg tgtgccgggc gttggtgagc tctatgcggt taccggccgt 901 ggtgatagtc cggccagctc tgccccgatc gccactagtg tgccgggcgt cggcgtgccg 961 ggcgtaggtg ttccgggcga gggtgttccg ggcgttggtg tgccgggcgt cggcgtgccg 1021 ggcgtgggtg ttccgggcgt aggtgtgccg ggcgagggtg tgccgggcgt gggcgtgccg 1081 ggcgtaggtg ttccgggcgt aggtgttccg ggcgtaggtg ttccgggtga aggcgtgccg 1141 ggcgttggtg tgccgggtgt gggcgtgccg ggcgggctgc tcgacatccc aacgaccgaa 1201 aacctgtatt ttcagggcgc catggttgat accttatcag gtttatcaag tgagcaaggt 1261 cagtccggtg atatgacaat tgaagaagat agtgctaccc atattaaatt ctcaaaacgt 1321 gatgaggacg gcaaagagtt agctggtgca actatggagt tgcgtgattc atctggtaaa 1381 actattagta catggatttc agatggacaa gtgaaagatt tctacctgta tccaggaaaa 1441 tatacatttg tcgaaaccgc agcaccagac ggttatgagg tagcaactgc tattaccttt 1501 acagttaatg agcaaggtca ggttactgta aatggcaaag caactaaagg tgacgctcat 1561 attggatccc ccgccaacct gaaggccctg gaggcccaga agcagaagga gcagagacag 1621 gccgccgagg agctggccaa cgccaagaag ctgaaggagc agctggagaa gggatcccat 1681 atgaagccgc tgcgtggtgc cgtgtttagc ctgcagaaac agcatcccga ctatcccgat 1741 atctatggcg cgattgatca gaatgggacc tatcaaaatg tgcgtaccgg cgaagatggt 1801 aaactgacct ttaagaatct gagcgatggc aaatatcgcc tgtttgaaaa tagcgaaccc 1861 gctggctata aaccggtgca gaataagccg attgtggcgt ttcagattgt gaatggcgaa 1921 gtgcgtgatg tgaccagcat tgtgccgcag gatattccgg ctacatatga atttaccaac 1981 ggtaaacatt atatcaccaa tgaaccgata ccgccgaaat aaggatccgg ctgctaacaa 2041 agcccgaagc ttaattagct gagcttggac tcctgttgat agatccagta atgacctcag 2101 aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt attggtgaga 2161 atccaagcca gcttggcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc 2221 actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt 2281 cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta 2341 aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc 2401 ctgatgaatg ctcatccgga atttcgtatg gcaatgaaag acggtgagct ggtgatatgg 2461 gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc 2521 tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg 2581 tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc 2641 tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac 2701 ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg 2761 ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt 2821 aatgaattac aacagtactg cgatgagtgg cagggcgggg cgtaattttt ttaaggcagt 2881 tattggtgcc cttaaacgcc tggggtaatg actctctagc ttgaggcatc aaataaaacg 2941 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct 3001 cctgagtagg acaaatccgc cctctagatt acgtgcagtc gatgataagc tgtcaaacat 3061 gagaattgtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct 3121 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 3181 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag 3241 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg 3301 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc 3361 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt 3421 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac 3481 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc 3541 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag 3601 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc 3661 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact 3721 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc 3781 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg 3841 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat 3901 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg 3961 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc 4021 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac 4081 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg 4141 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac 4201 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcaccattc 4261 gatggtgtcg gaatttcggg cagcgttggg tcctggccac gggtgcgcat gatctagagc 4321 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg 4381 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg 4441 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat 4501 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg 4561 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc 4621 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 4681 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 4741 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 4801 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 4861 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 4921 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 4981 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 5041 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 5101 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 5161 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 5221 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 5281 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 5341 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 5401 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 5461 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 5521 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 5581 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 5641 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 5701 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 5761 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 5821 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 5881 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 5941 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 6001 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 6061 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 6121 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 6181 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 6241 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 6301 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 6361 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 6421 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 6481 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgac //
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