Plasmid Preparation:Article Title: Recombinant Photo-tagging Enables Fluorescent Labelling of Biomolecules and Visualization of Liquid–Liquid Phase Separation
Article Snippet: Costar® 96-well UV-transparent microplates and black fluorescence microplates were purchased from Corning Incorporated. .. Mj RibK plasmid was a generous gift from Dr. Amrita Hazra (Whytamin Lab IISER Pune (India). p ET-PhoCl1-6xHis was a gift from Robert Campbell (Addgene plasmid #164033; http://n2t.net/addgene:164033 ; RRID: Addgene_164033). pET28-MBP-super TEV protease was a gift from Mark Howarth (Addgene plasmid # 171782 ; http://n2t.net/addgene:171782 ; RRID:Addgene_171782). .. V48 ELP was a gift from Andrew MacKay (Addgene plasmid # 68395 ; http://n2t.net/addgene:68395 ; RRID:Addgene_68395).
Article Title: SpyMask enables combinatorial assembly of bispecific binders.
Article Snippet: .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888, Addgene plasmid ID 133450)28, pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453)28, pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448)60, pET28-MBP-super TEV protease (Addgene plasmid ID 171782)26, pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His6–SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975, Addgene plasmid ID 216280)84, and pET28a-SnoopTagSpyTag-(AffiHER2)3 (GenBank Accession no. KU296976, Addgene plasmid ID 216281)33 have been described. pET28a-SUMO-SpyTag003 (N-terminal His6 tag-SUMOprotein-SpyTag003) was cloned previously by Dr. Irsyad Khairil Anuar (University of Oxford) (GenBank Accession no. PP341235, Addgene plasmid ID 216282). pDEST14-SpyCatcher003TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His6 tagSpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBankAccession no. PP341217, Addgeneplasmid ID 216283)wasderived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887, Addgene plasmid ID 133447) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK)28 byGibson assembly. pDEST14-SpyCatcher003-(GSG)3-SpyCatcher003TEVs-SpyTag003DA [DoubleCatcher;N-terminalHis6 tag-TEVprotease cleavage site-two SpyCatcher003 moieties connected by a (GSG)3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank Accession no. PP341218, Addgene plasmid ID 216284] was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. TheGly/Ser spacer in this constructwas then replacedwith anα-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK)46 and this construct was termed DoubleCatcher H-Lock (GenBank Accession no. PP341219, Addgene plasmid ID 216285).
Article Title: SpyCombinator Assembly of Bispecific Binders
Article Snippet: Constructs were cloned by PCR methods using Q5 High-Fidelity Polymerase (New England Biolabs) and fragments were assembled by Gibson assembly. .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888 , Addgene plasmid ID 133450), pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453) , pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448) , pET28-MBP-super TEV protease (Addgene plasmid ID 171782) , pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His 6 –SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975 ) (Addgene deposition in progress) , and pET28a-SnoopTag-SpyTag-(AffiHER2) 3 (GenBank accession no. KU296976 ) (Addgene deposition in progress) ( ) have been described. pET28a-SUMO-SpyTag003 (N-terminal His 6 tag-SUMO protein-SpyTag003) was cloned previously by Irsyad Khairil Anuar (University of Oxford) (GenBank and Addgene deposition in progress). pDEST14-SpyCatcher003-TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His 6 tag-SpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBank and Addgene deposition in progress) was derived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887 , Addgene plasmid ID 133447) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK) ( ) by Gibson assembly. pDEST14-SpyCatcher003-(GSG) 3 -SpyCatcher003-TEVs-SpyTag003DA (DoubleCatcher; N-terminal His 6 tag-TEV protease cleavage site-two SpyCatcher003 moieties connected by a (GSG) 3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank and Addgene deposition in progress) was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. The Gly/Ser spacer in this construct was then replaced with an ⍺-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK) , and this construct was termed DoubleCatcher H-Lock (GenBank and Addgene deposition in progress).
Article Title: SpyMask enables combinatorial assembly of bispecific binders
Article Snippet: .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888 , Addgene plasmid ID 133450 ) , pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453 ) , pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448 ) , pET28-MBP-super TEV protease (Addgene plasmid ID 171782 ) , pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His 6 –SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975 , Addgene plasmid ID 216280 ) , and pET28a-SnoopTag-SpyTag-(AffiHER2) 3 (GenBank Accession no. KU296976 , Addgene plasmid ID 216281 ) have been described. pET28a-SUMO-SpyTag003 (N-terminal His 6 tag-SUMO protein-SpyTag003) was cloned previously by Dr. Irsyad Khairil Anuar (University of Oxford) (GenBank Accession no. PP341235 , Addgene plasmid ID 216282 ). pDEST14-SpyCatcher003-TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His 6 tag-SpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBank Accession no. PP341217 , Addgene plasmid ID 216283 ) was derived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887 , Addgene plasmid ID 133447 ) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK) by Gibson assembly. pDEST14-SpyCatcher003-(GSG) 3 -SpyCatcher003-TEVs-SpyTag003DA [DoubleCatcher; N-terminal His 6 tag-TEV protease cleavage site-two SpyCatcher003 moieties connected by a (GSG) 3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank Accession no. PP341218 , Addgene plasmid ID 216284 ] was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. The Gly/Ser spacer in this construct was then replaced with an α-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK) and this construct was termed DoubleCatcher H-Lock (GenBank Accession no. PP341219 , Addgene plasmid ID 216285 ).
Construct:Article Title: SpyMask enables combinatorial assembly of bispecific binders.
Article Snippet: .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888, Addgene plasmid ID 133450)28, pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453)28, pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448)60, pET28-MBP-super TEV protease (Addgene plasmid ID 171782)26, pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His6–SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975, Addgene plasmid ID 216280)84, and pET28a-SnoopTagSpyTag-(AffiHER2)3 (GenBank Accession no. KU296976, Addgene plasmid ID 216281)33 have been described. pET28a-SUMO-SpyTag003 (N-terminal His6 tag-SUMOprotein-SpyTag003) was cloned previously by Dr. Irsyad Khairil Anuar (University of Oxford) (GenBank Accession no. PP341235, Addgene plasmid ID 216282). pDEST14-SpyCatcher003TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His6 tagSpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBankAccession no. PP341217, Addgeneplasmid ID 216283)wasderived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887, Addgene plasmid ID 133447) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK)28 byGibson assembly. pDEST14-SpyCatcher003-(GSG)3-SpyCatcher003TEVs-SpyTag003DA [DoubleCatcher;N-terminalHis6 tag-TEVprotease cleavage site-two SpyCatcher003 moieties connected by a (GSG)3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank Accession no. PP341218, Addgene plasmid ID 216284] was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. TheGly/Ser spacer in this constructwas then replacedwith anα-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK)46 and this construct was termed DoubleCatcher H-Lock (GenBank Accession no. PP341219, Addgene plasmid ID 216285).
Article Title: SpyCombinator Assembly of Bispecific Binders
Article Snippet: Constructs were cloned by PCR methods using Q5 High-Fidelity Polymerase (New England Biolabs) and fragments were assembled by Gibson assembly. .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888 , Addgene plasmid ID 133450), pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453) , pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448) , pET28-MBP-super TEV protease (Addgene plasmid ID 171782) , pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His 6 –SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975 ) (Addgene deposition in progress) , and pET28a-SnoopTag-SpyTag-(AffiHER2) 3 (GenBank accession no. KU296976 ) (Addgene deposition in progress) ( ) have been described. pET28a-SUMO-SpyTag003 (N-terminal His 6 tag-SUMO protein-SpyTag003) was cloned previously by Irsyad Khairil Anuar (University of Oxford) (GenBank and Addgene deposition in progress). pDEST14-SpyCatcher003-TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His 6 tag-SpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBank and Addgene deposition in progress) was derived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887 , Addgene plasmid ID 133447) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK) ( ) by Gibson assembly. pDEST14-SpyCatcher003-(GSG) 3 -SpyCatcher003-TEVs-SpyTag003DA (DoubleCatcher; N-terminal His 6 tag-TEV protease cleavage site-two SpyCatcher003 moieties connected by a (GSG) 3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank and Addgene deposition in progress) was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. The Gly/Ser spacer in this construct was then replaced with an ⍺-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK) , and this construct was termed DoubleCatcher H-Lock (GenBank and Addgene deposition in progress).
Article Title: SpyMask enables combinatorial assembly of bispecific binders
Article Snippet: .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888 , Addgene plasmid ID 133450 ) , pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453 ) , pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448 ) , pET28-MBP-super TEV protease (Addgene plasmid ID 171782 ) , pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His 6 –SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975 , Addgene plasmid ID 216280 ) , and pET28a-SnoopTag-SpyTag-(AffiHER2) 3 (GenBank Accession no. KU296976 , Addgene plasmid ID 216281 ) have been described. pET28a-SUMO-SpyTag003 (N-terminal His 6 tag-SUMO protein-SpyTag003) was cloned previously by Dr. Irsyad Khairil Anuar (University of Oxford) (GenBank Accession no. PP341235 , Addgene plasmid ID 216282 ). pDEST14-SpyCatcher003-TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His 6 tag-SpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBank Accession no. PP341217 , Addgene plasmid ID 216283 ) was derived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887 , Addgene plasmid ID 133447 ) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK) by Gibson assembly. pDEST14-SpyCatcher003-(GSG) 3 -SpyCatcher003-TEVs-SpyTag003DA [DoubleCatcher; N-terminal His 6 tag-TEV protease cleavage site-two SpyCatcher003 moieties connected by a (GSG) 3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank Accession no. PP341218 , Addgene plasmid ID 216284 ] was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. The Gly/Ser spacer in this construct was then replaced with an α-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK) and this construct was termed DoubleCatcher H-Lock (GenBank Accession no. PP341219 , Addgene plasmid ID 216285 ).
Sequencing:Article Title: SpyMask enables combinatorial assembly of bispecific binders.
Article Snippet: .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888, Addgene plasmid ID 133450)28, pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453)28, pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448)60, pET28-MBP-super TEV protease (Addgene plasmid ID 171782)26, pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His6–SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975, Addgene plasmid ID 216280)84, and pET28a-SnoopTagSpyTag-(AffiHER2)3 (GenBank Accession no. KU296976, Addgene plasmid ID 216281)33 have been described. pET28a-SUMO-SpyTag003 (N-terminal His6 tag-SUMOprotein-SpyTag003) was cloned previously by Dr. Irsyad Khairil Anuar (University of Oxford) (GenBank Accession no. PP341235, Addgene plasmid ID 216282). pDEST14-SpyCatcher003TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His6 tagSpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBankAccession no. PP341217, Addgeneplasmid ID 216283)wasderived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887, Addgene plasmid ID 133447) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK)28 byGibson assembly. pDEST14-SpyCatcher003-(GSG)3-SpyCatcher003TEVs-SpyTag003DA [DoubleCatcher;N-terminalHis6 tag-TEVprotease cleavage site-two SpyCatcher003 moieties connected by a (GSG)3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank Accession no. PP341218, Addgene plasmid ID 216284] was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. TheGly/Ser spacer in this constructwas then replacedwith anα-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK)46 and this construct was termed DoubleCatcher H-Lock (GenBank Accession no. PP341219, Addgene plasmid ID 216285).
Article Title: SpyCombinator Assembly of Bispecific Binders
Article Snippet: Constructs were cloned by PCR methods using Q5 High-Fidelity Polymerase (New England Biolabs) and fragments were assembled by Gibson assembly. .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888 , Addgene plasmid ID 133450), pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453) , pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448) , pET28-MBP-super TEV protease (Addgene plasmid ID 171782) , pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His 6 –SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975 ) (Addgene deposition in progress) , and pET28a-SnoopTag-SpyTag-(AffiHER2) 3 (GenBank accession no. KU296976 ) (Addgene deposition in progress) ( ) have been described. pET28a-SUMO-SpyTag003 (N-terminal His 6 tag-SUMO protein-SpyTag003) was cloned previously by Irsyad Khairil Anuar (University of Oxford) (GenBank and Addgene deposition in progress). pDEST14-SpyCatcher003-TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His 6 tag-SpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBank and Addgene deposition in progress) was derived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887 , Addgene plasmid ID 133447) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK) ( ) by Gibson assembly. pDEST14-SpyCatcher003-(GSG) 3 -SpyCatcher003-TEVs-SpyTag003DA (DoubleCatcher; N-terminal His 6 tag-TEV protease cleavage site-two SpyCatcher003 moieties connected by a (GSG) 3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank and Addgene deposition in progress) was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. The Gly/Ser spacer in this construct was then replaced with an ⍺-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK) , and this construct was termed DoubleCatcher H-Lock (GenBank and Addgene deposition in progress).
Article Title: SpyMask enables combinatorial assembly of bispecific binders
Article Snippet: .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888 , Addgene plasmid ID 133450 ) , pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453 ) , pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448 ) , pET28-MBP-super TEV protease (Addgene plasmid ID 171782 ) , pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His 6 –SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975 , Addgene plasmid ID 216280 ) , and pET28a-SnoopTag-SpyTag-(AffiHER2) 3 (GenBank Accession no. KU296976 , Addgene plasmid ID 216281 ) have been described. pET28a-SUMO-SpyTag003 (N-terminal His 6 tag-SUMO protein-SpyTag003) was cloned previously by Dr. Irsyad Khairil Anuar (University of Oxford) (GenBank Accession no. PP341235 , Addgene plasmid ID 216282 ). pDEST14-SpyCatcher003-TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His 6 tag-SpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBank Accession no. PP341217 , Addgene plasmid ID 216283 ) was derived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887 , Addgene plasmid ID 133447 ) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK) by Gibson assembly. pDEST14-SpyCatcher003-(GSG) 3 -SpyCatcher003-TEVs-SpyTag003DA [DoubleCatcher; N-terminal His 6 tag-TEV protease cleavage site-two SpyCatcher003 moieties connected by a (GSG) 3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank Accession no. PP341218 , Addgene plasmid ID 216284 ] was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. The Gly/Ser spacer in this construct was then replaced with an α-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK) and this construct was termed DoubleCatcher H-Lock (GenBank Accession no. PP341219 , Addgene plasmid ID 216285 ).
Clone Assay:Article Title: SpyMask enables combinatorial assembly of bispecific binders.
Article Snippet: .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888, Addgene plasmid ID 133450)28, pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453)28, pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448)60, pET28-MBP-super TEV protease (Addgene plasmid ID 171782)26, pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His6–SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975, Addgene plasmid ID 216280)84, and pET28a-SnoopTagSpyTag-(AffiHER2)3 (GenBank Accession no. KU296976, Addgene plasmid ID 216281)33 have been described. pET28a-SUMO-SpyTag003 (N-terminal His6 tag-SUMOprotein-SpyTag003) was cloned previously by Dr. Irsyad Khairil Anuar (University of Oxford) (GenBank Accession no. PP341235, Addgene plasmid ID 216282). pDEST14-SpyCatcher003TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His6 tagSpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBankAccession no. PP341217, Addgeneplasmid ID 216283)wasderived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887, Addgene plasmid ID 133447) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK)28 byGibson assembly. pDEST14-SpyCatcher003-(GSG)3-SpyCatcher003TEVs-SpyTag003DA [DoubleCatcher;N-terminalHis6 tag-TEVprotease cleavage site-two SpyCatcher003 moieties connected by a (GSG)3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank Accession no. PP341218, Addgene plasmid ID 216284] was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. TheGly/Ser spacer in this constructwas then replacedwith anα-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK)46 and this construct was termed DoubleCatcher H-Lock (GenBank Accession no. PP341219, Addgene plasmid ID 216285).
Article Title: SpyCombinator Assembly of Bispecific Binders
Article Snippet: Constructs were cloned by PCR methods using Q5 High-Fidelity Polymerase (New England Biolabs) and fragments were assembled by Gibson assembly. .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888 , Addgene plasmid ID 133450), pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453) , pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448) , pET28-MBP-super TEV protease (Addgene plasmid ID 171782) , pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His 6 –SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975 ) (Addgene deposition in progress) , and pET28a-SnoopTag-SpyTag-(AffiHER2) 3 (GenBank accession no. KU296976 ) (Addgene deposition in progress) ( ) have been described. pET28a-SUMO-SpyTag003 (N-terminal His 6 tag-SUMO protein-SpyTag003) was cloned previously by Irsyad Khairil Anuar (University of Oxford) (GenBank and Addgene deposition in progress). pDEST14-SpyCatcher003-TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His 6 tag-SpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBank and Addgene deposition in progress) was derived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887 , Addgene plasmid ID 133447) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK) ( ) by Gibson assembly. pDEST14-SpyCatcher003-(GSG) 3 -SpyCatcher003-TEVs-SpyTag003DA (DoubleCatcher; N-terminal His 6 tag-TEV protease cleavage site-two SpyCatcher003 moieties connected by a (GSG) 3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank and Addgene deposition in progress) was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. The Gly/Ser spacer in this construct was then replaced with an ⍺-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK) , and this construct was termed DoubleCatcher H-Lock (GenBank and Addgene deposition in progress).
Article Title: SpyMask enables combinatorial assembly of bispecific binders
Article Snippet: .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888 , Addgene plasmid ID 133450 ) , pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453 ) , pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448 ) , pET28-MBP-super TEV protease (Addgene plasmid ID 171782 ) , pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His 6 –SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975 , Addgene plasmid ID 216280 ) , and pET28a-SnoopTag-SpyTag-(AffiHER2) 3 (GenBank Accession no. KU296976 , Addgene plasmid ID 216281 ) have been described. pET28a-SUMO-SpyTag003 (N-terminal His 6 tag-SUMO protein-SpyTag003) was cloned previously by Dr. Irsyad Khairil Anuar (University of Oxford) (GenBank Accession no. PP341235 , Addgene plasmid ID 216282 ). pDEST14-SpyCatcher003-TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His 6 tag-SpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBank Accession no. PP341217 , Addgene plasmid ID 216283 ) was derived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887 , Addgene plasmid ID 133447 ) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK) by Gibson assembly. pDEST14-SpyCatcher003-(GSG) 3 -SpyCatcher003-TEVs-SpyTag003DA [DoubleCatcher; N-terminal His 6 tag-TEV protease cleavage site-two SpyCatcher003 moieties connected by a (GSG) 3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank Accession no. PP341218 , Addgene plasmid ID 216284 ] was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. The Gly/Ser spacer in this construct was then replaced with an α-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK) and this construct was termed DoubleCatcher H-Lock (GenBank Accession no. PP341219 , Addgene plasmid ID 216285 ).
Synthesized:Article Title: SpyMask enables combinatorial assembly of bispecific binders.
Article Snippet: .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888, Addgene plasmid ID 133450)28, pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453)28, pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448)60, pET28-MBP-super TEV protease (Addgene plasmid ID 171782)26, pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His6–SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975, Addgene plasmid ID 216280)84, and pET28a-SnoopTagSpyTag-(AffiHER2)3 (GenBank Accession no. KU296976, Addgene plasmid ID 216281)33 have been described. pET28a-SUMO-SpyTag003 (N-terminal His6 tag-SUMOprotein-SpyTag003) was cloned previously by Dr. Irsyad Khairil Anuar (University of Oxford) (GenBank Accession no. PP341235, Addgene plasmid ID 216282). pDEST14-SpyCatcher003TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His6 tagSpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBankAccession no. PP341217, Addgeneplasmid ID 216283)wasderived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887, Addgene plasmid ID 133447) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK)28 byGibson assembly. pDEST14-SpyCatcher003-(GSG)3-SpyCatcher003TEVs-SpyTag003DA [DoubleCatcher;N-terminalHis6 tag-TEVprotease cleavage site-two SpyCatcher003 moieties connected by a (GSG)3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank Accession no. PP341218, Addgene plasmid ID 216284] was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. TheGly/Ser spacer in this constructwas then replacedwith anα-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK)46 and this construct was termed DoubleCatcher H-Lock (GenBank Accession no. PP341219, Addgene plasmid ID 216285).
Article Title: SpyCombinator Assembly of Bispecific Binders
Article Snippet: Constructs were cloned by PCR methods using Q5 High-Fidelity Polymerase (New England Biolabs) and fragments were assembled by Gibson assembly. .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888 , Addgene plasmid ID 133450), pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453) , pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448) , pET28-MBP-super TEV protease (Addgene plasmid ID 171782) , pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His 6 –SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975 ) (Addgene deposition in progress) , and pET28a-SnoopTag-SpyTag-(AffiHER2) 3 (GenBank accession no. KU296976 ) (Addgene deposition in progress) ( ) have been described. pET28a-SUMO-SpyTag003 (N-terminal His 6 tag-SUMO protein-SpyTag003) was cloned previously by Irsyad Khairil Anuar (University of Oxford) (GenBank and Addgene deposition in progress). pDEST14-SpyCatcher003-TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His 6 tag-SpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBank and Addgene deposition in progress) was derived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887 , Addgene plasmid ID 133447) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK) ( ) by Gibson assembly. pDEST14-SpyCatcher003-(GSG) 3 -SpyCatcher003-TEVs-SpyTag003DA (DoubleCatcher; N-terminal His 6 tag-TEV protease cleavage site-two SpyCatcher003 moieties connected by a (GSG) 3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank and Addgene deposition in progress) was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. The Gly/Ser spacer in this construct was then replaced with an ⍺-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK) , and this construct was termed DoubleCatcher H-Lock (GenBank and Addgene deposition in progress).
Article Title: SpyMask enables combinatorial assembly of bispecific binders
Article Snippet: .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888 , Addgene plasmid ID 133450 ) , pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453 ) , pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448 ) , pET28-MBP-super TEV protease (Addgene plasmid ID 171782 ) , pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His 6 –SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975 , Addgene plasmid ID 216280 ) , and pET28a-SnoopTag-SpyTag-(AffiHER2) 3 (GenBank Accession no. KU296976 , Addgene plasmid ID 216281 ) have been described. pET28a-SUMO-SpyTag003 (N-terminal His 6 tag-SUMO protein-SpyTag003) was cloned previously by Dr. Irsyad Khairil Anuar (University of Oxford) (GenBank Accession no. PP341235 , Addgene plasmid ID 216282 ). pDEST14-SpyCatcher003-TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His 6 tag-SpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBank Accession no. PP341217 , Addgene plasmid ID 216283 ) was derived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887 , Addgene plasmid ID 133447 ) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK) by Gibson assembly. pDEST14-SpyCatcher003-(GSG) 3 -SpyCatcher003-TEVs-SpyTag003DA [DoubleCatcher; N-terminal His 6 tag-TEV protease cleavage site-two SpyCatcher003 moieties connected by a (GSG) 3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank Accession no. PP341218 , Addgene plasmid ID 216284 ] was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. The Gly/Ser spacer in this construct was then replaced with an α-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK) and this construct was termed DoubleCatcher H-Lock (GenBank Accession no. PP341219 , Addgene plasmid ID 216285 ).
Derivative Assay:Article Title: SpyCombinator Assembly of Bispecific Binders
Article Snippet: Constructs were cloned by PCR methods using Q5 High-Fidelity Polymerase (New England Biolabs) and fragments were assembled by Gibson assembly. .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888 , Addgene plasmid ID 133450), pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453) , pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448) , pET28-MBP-super TEV protease (Addgene plasmid ID 171782) , pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His 6 –SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975 ) (Addgene deposition in progress) , and pET28a-SnoopTag-SpyTag-(AffiHER2) 3 (GenBank accession no. KU296976 ) (Addgene deposition in progress) ( ) have been described. pET28a-SUMO-SpyTag003 (N-terminal His 6 tag-SUMO protein-SpyTag003) was cloned previously by Irsyad Khairil Anuar (University of Oxford) (GenBank and Addgene deposition in progress). pDEST14-SpyCatcher003-TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His 6 tag-SpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBank and Addgene deposition in progress) was derived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887 , Addgene plasmid ID 133447) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK) ( ) by Gibson assembly. pDEST14-SpyCatcher003-(GSG) 3 -SpyCatcher003-TEVs-SpyTag003DA (DoubleCatcher; N-terminal His 6 tag-TEV protease cleavage site-two SpyCatcher003 moieties connected by a (GSG) 3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank and Addgene deposition in progress) was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. The Gly/Ser spacer in this construct was then replaced with an ⍺-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK) , and this construct was termed DoubleCatcher H-Lock (GenBank and Addgene deposition in progress).
Article Title: SpyMask enables combinatorial assembly of bispecific binders
Article Snippet: .. All constructs were validated by Sanger Sequencing. pET28a-SpyTag003-MBP (GenBank Accession no. MN433888 , Addgene plasmid ID 133450 ) , pET28-SpyTag003-mClover3 (Addgene plasmid ID 133453 ) , pDEST14-SpyCatcher003 S49C (Addgene plasmid ID 133448 ) , pET28-MBP-super TEV protease (Addgene plasmid ID 171782 ) , pET28a-SnoopTag-AffiHER2-SpyTag (N-terminal His 6 –SnoopTag–anti-HER2 Affibody–SpyTag) (GenBank accession no. KU296975 , Addgene plasmid ID 216280 ) , and pET28a-SnoopTag-SpyTag-(AffiHER2) 3 (GenBank Accession no. KU296976 , Addgene plasmid ID 216281 ) have been described. pET28a-SUMO-SpyTag003 (N-terminal His 6 tag-SUMO protein-SpyTag003) was cloned previously by Dr. Irsyad Khairil Anuar (University of Oxford) (GenBank Accession no. PP341235 , Addgene plasmid ID 216282 ). pDEST14-SpyCatcher003-TEVs-SpyTag003DA (‘Masked SpyCatcher0003’; N-terminal His 6 tag-SpyCatcher003-TEV protease cleavage site-SpyTag003 D117A, GenBank Accession no. PP341217 , Addgene plasmid ID 216283 ) was derived from pDEST14-SpyCatcher003 (GenBank Accession no. MN433887 , Addgene plasmid ID 133447 ) by incorporating the TEV protease cleavage site (ENLYFQ/G) and SpyTag003 D117A (RGVPHIVMVAAYKRYK) by Gibson assembly. pDEST14-SpyCatcher003-(GSG) 3 -SpyCatcher003-TEVs-SpyTag003DA [DoubleCatcher; N-terminal His 6 tag-TEV protease cleavage site-two SpyCatcher003 moieties connected by a (GSG) 3 spacer-TEV protease cleavage site-SpyTag003 DA, GenBank Accession no. PP341218 , Addgene plasmid ID 216284 ] was synthesized as a gene fragment (Integrated DNA Technologies) and inserted into pDEST14. .. The Gly/Ser spacer in this construct was then replaced with an α-helical spacer (sequence PANLKALEAQKQKEQRQAAEELANAKKLKEQLEK) and this construct was termed DoubleCatcher H-Lock (GenBank Accession no. PP341219 , Addgene plasmid ID 216285 ).
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