SBB09 Construct45

Construction of {<N.fliD>} (replaced below)

PCR Omv044F/Omv045R on MG1655 genomic DNA  (488bp, gp=A)
PCR Omv045F/Omv044R on MG1655 genomic DNA  (178bp, small frag cleanup=B)
----
PCR Omv044F/Omv044R on A + B  (643bp, EcoRI/BamHI)
Sub into pBca9495AC-Bca1144#5  (EcoRI/BamHI, L)
Product is pBca9495AC-M10000  {<N.fliD>}
----
Omv044F  Forward Bglbricking of {<N.fliD>}  ccagtGAATTCatgAGATCTgcaagtatttcatcgctggg
Omv044R  Reverse Bglbricking of {<N.fliD!}  gcagtGGATCCaaaactttgtagcgcatcatcaccgc
Omv045F  Forward Removing BamHI site in {<N.fliD>}  cgttaagcggCatccgtgatgcc
Omv045R  Reverse Removing BamHI site in {<N.fliD>}  ggcatcacggatGccgcttaacg

 JCA:  I think you want to make this as an N-terminal display system.  So, you'll want a
 {<part!>} version instead of {<part>}.  So, I've added a stop codon to Omv044R below:

Construction of {<N.fliD!}

PCR Omv044F/Omv045R on MG1655 genomic DNA  (488bp, gp=A)
PCR Omv045F/Omv044R on MG1655 genomic DNA  (178bp, small frag cleanup=B)
----
PCR Omv044F/Omv044R on A + B  (643bp, EcoRI/BamHI)
Sub into pBca9495AC-Bca1144#5  (EcoRI/BamHI, L)
Product is pBca9495AC-M10000  {<N.fliD!}
----
Omv044F  Forward Bglbricking of {<N.fliD>}  ccagtGAATTCatgAGATCTgcaagtatttcatcgctggg
Omv044R  Reverse Bglbricking of {<N.fliD>}  gcagtGGATCCttaaaaactttgtagcgcatcatcaccgc
Omv045F  Forward Removing BamHI site in {<N.fliD>}  cgttaagcggCatccgtgatgcc
Omv045R  Reverse Removing BamHI site in {<N.fliD>}  ggcatcacggatGccgcttaacg

Construction of {<C.fliD>}

PCR Omv046F/Omv046R on MG1655 genomic DNA  (781, EcoRI/BamHI)
Sub into pBca9495KC-Bca1144#5  (EcoRI/BamHI, 2974+910, L)
Product is pBca9495KC-M10001  {<C.fliD>}
----
Omv046F  Forward Bglbricking of {<C.fliD>}  ccagtGAATTCatgAGATCTgttgccgcccagaatgcg
Omv046R  Reverse Bglbricking of {<C.fliD>}  gcagtGGATCCcttggaattactgttgttttcg

Bglbricking of {<linker1>}

Wobble Omv047F/Omv047R         (115 bp, EcoRI/BamHI)
Sub into pBca9495CA-Bca1144#5   (EcoRI/BamHI, 3039+910, L)
Product is pBca9495CA-M10002     {<linker1>}
--------------------------
Omv047F   Forward construction of {<linker1>} basic part
ccataGAATTCatgAGATCTtacttcgatgtctggggcgcagggaccacggtcaccgtctcctcaggcggag
Omv047R   Reverse construction of {<linker1>} basic part
ctgatGGATCCtcctcctccagaaccacctccacctgaaccgcctccgcctgaggagacggtgac

Bglbricking of {<linker2>}

Wobble Omv048F/Omv048R         (112 bp, EcoRI/BamHI)
Sub into pBca9495CA-Bca1144#5  (EcoRI/BamHI, 3039+910, L)
Product is pBca9495CA-M10003     {<linker2>}
---------------------------
Omv048F   Forward construction of {<linker2>} basic part
ccataGAATTCatgAGATCTtggacgttcggtggaggcaccaagctggaaatcaaacgtgcggccgctc
Omv048R   Reverse construction of {<linker2>} basic part
ctgatGGATCCacggccttcaatgccgtgatggtgatggtggtgagcggccgcacgtttgatttc

Note: These parts will be assembled using double antibiotic assembly to make a circularly permuted N-terminal displayer part in a KC vector (C.fliD will be assembled with the linker before being assembled with N.fliD). I currently have not included a stop codon at the end of the contruct, but I don't know if I should add one (it didn't seem like it from the circular permutation tutorial).

Alternate Constructions using SOEing Instead of Assembly

PCR Omv046F/Omv051R on pBca1256-M10001  (819bp, gp=A)
PCR Omv051F/Omv044R on pBca1256-M10000  (674bp, gp=B)
----
PCR Omv046F/Omv044R on A + B  (1471bp, EcoRI/BamHI)
Sub into pBca9495KC-Bca1144#5 (EcoRI/BamHI,2974+910, L)
Product in pBca9495KC-M10058  {<C.fliD.linker1.N.fliD!}
----
Omv046F  Forward Bglbricking of {<C.fliD>}  
ccagtGAATTCatgAGATCTgttgccgcccagaatgcg
Omv051R  Reverse on {<C.fliD>} for SOEing linker1
ctccgcctgaggagacggtgaccgtggtccctgcgccccagacatcgaagtaCTTGGAATTACTGTTGTTTTCG
Omv051F  Forward on {<N.fliD!} for SOEing linker1
gtcaccgtctcctcaggcggaggcggttcaggtggaggtggttctggaggaggaGCAAGTATTTCATCGCTGGG
Omv044R  Reverse Bglbricking of {<N.fliD!}  
gcagtGGATCCaaaactttgtagcgcatcatcaccgc

PCR Omv046F/Omv052R on pBca1256-M10001  (816bp, gp=C)
PCR Omv052F/Omv044R on pBca1256-M10000  (674bp, gp=D)
----
PCR Omv046F/Omv044R on C + D  (1468bp, EcoRI/BamHI)
Sub into pBca9495KC-Bca1144#5 (EcoRI/BamHI,2974+910, L)
Product in pBca9495KC-M10059  {<C.fliD.linker1.N.fliD!}
----
Omv046F  Forward Bglbricking of {<C.fliD>}  
ccagtGAATTCatgAGATCTgttgccgcccagaatgcg
Omv052R  Reverse on {<C.fliD>} for SOEing linker2
gagcggccgcacgtttgatttccagcttggtgcctccaccgaacgtccaCTTGGAATTACTGTTGTTTTCG
Omv052F  Forward on {<N.fliD!} for SOEing linker2
gaaatcaaacgtgcggccgctcaccaccatcaccatcacggcattgaaggccgtGCAAGTATTTCATCGCTGGG
Omv044R  Reverse Bglbricking of {<N.fliD!}  
gcagtGGATCCaaaactttgtagcgcatcatcaccgc