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First use BsaI to ligate start/finish half edges into the HPP vector

First use BsaI to ligate start/finish half edges into the HPP vector. S. F. RI- BsaI - - - - GTGG - BsmBI - Pst. RI- BsmBI - GTGG - - BsaI - Pst. Chlor. Chlor. RI – BsaI – CCDB – BsaI - Pst. Amp. F. S. RI- - - - GTGG - BsmBI.

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First use BsaI to ligate start/finish half edges into the HPP vector

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  1. First use BsaI to ligate start/finish half edges into the HPP vector S F RI-BsaI- - - - GTGG- BsmBI - Pst RI- BsmBI- GTGG - - BsaI- Pst Chlor Chlor RI – BsaI– CCDB – BsaI -Pst Amp F S RI- - - - GTGG- BsmBI BsmBI- GTGG - Pst Amp Use GGS to build a path with half edges then using BsaI, BsmBI, and ligase to insert into HPP Vector S F 1 1 2 2 BsmBI-GTGG - - - -BsaI BsaI - - - - - GTGG- BsmBI Chlor Chlor 1 F S 1 2 F S 2 RI- - - - - - - - - Amp

  2. First Cut with BsaI S F RI-BsaI- - - - GTGG- BsmBI BsmBI- GTGG - - BsaI- Pst Chlor Chlor RI – BsaI– CCDB – BsaI -Pst Amp F S - - - GTGG- BsmBI BsmBI- GTGG - Chlor Chlor RI – Pst Amp F S RI- - - - GTGG- BsmBI BsmBI- GTGG - Pst Amp

  3. Second Build graph using GGS S F 1 1 2 2 BsmBI-GTGG - - - -BsaI BsaI - - - - - GTGG- BsmBI Chlor Chlor Digest the HPP vector and the graph vector with BsmBI and insert graph into Vector 1 F S 1 2 2 BsmBI-GTGG- - -BsaI BsaI- - - - - - GTGG- Bs Chlor Chlor S F S RI- - - - GTGG- BsmBI BsmBI- GTGG - Pst Amp 1 F S 1 2 F S 2 RI- - - - - - - - - -Pst Amp

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