Difference between revisions of "Part:BBa K1607010:Design"

Revision as of 05:34, 24 August 2015

The coding sequence of the SCFV of anti-p185her2/neu antibody chA21

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 370
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

The termination codon:

   Since it was originally designed for 3'his-tag vectors, this coding sequence does not have a termination codon.

Source

This coding sequence was synthesized by LCR assmebly using 36 oilgos

AATTCCGattgtgtcc

ggacacaatCGGAATTCATGGGTTCTACTCAAG

CATATCAGTACCAGTACAAACTTGAGTAGAACCCATG

TTTGTACTGGTACTGATATGAAGTTGAGATTGCCAG

AATGAGTTTCTGGAGAAGCTGGCAATCTCAACTT

CTTCTCCAGAAACTCATTTGGATATGTTGAGACATTT

CTTGACAACCTTGGTACAAATGTCTCAACATATCCA

GTACCAAGGTTGTCAAGTTGTTCAAGGTAACTTGG

TGGCAAGTAAGTCAATTCCAAGTTACCTTGAACAA

AATTGACTTACTTGCCAACTAACGCTTCTTTGTC

TCTTGAATATCTTGCAAAAAAGACAAAGAAGCGTTAGT

TTTTTTGCAAGATATTCAAGAAGTTCAAGGTTACGTTT

CTTGGTTATGAGCAATCAAAACGTAACCTTGAACT

TGATTGCTCATAACCAAGTTAGACAAGTTCCATTGC

CTCTAACAATTCTCAATCTTTGCAATGGAACTTGTCTAA

AAAGATTGAGAATTGTTAGAGGTACTCAATTGTTTGAAGAT

CAGCCAAAGCGTAGTTATCTTCAAACAATTGAGTAC

AACTACGCTTTGGCTGTTTTGGATAACGGTGATC

GGAGTAGTGTTGTTCAATGGATCACCGTTATCCAAAA

CATTGAACAACACTACTCCAGTTACTGGTGCTTCT

CTCTCAAACCACCTGGAGAAGCACCAGTAACT

CCAGGTGGTTTGAGAGAATTGCAATTGAGATCTTT

CCCTTCAAAATTTCAGTCAAAGATCTCAATTGCAATT

GACTGAAATTTTGAAGGGTGGTGTTTTGATTCAAAG

ACACAATTGTGGGTTTCTTTGAATCAAAACACCA

AAACCCACAATTGTGTTACCAAGATACTATTTTGTGG

TGTTCTTATGAAAAATATCCTTCCACAAAATAGTATCTTGGTA

AAGGATATTTTTCATAAGAACAACCAATTGGCTTTGAC

GATCTGTTAGTATCAATCAAAGTCAAAGCCAATTGGT

TTTGATTGATACTAACAGATCCAGAGCTTGTCATCCA

CTTACACATTGGAGAACATGGATGACAAGCTCTG

TGTTCTCCAATGTGTAAGGGTTCCAGATGTTGGG

CAATCTTCAGAAGATTCACCCCAACATCTGGAACC

GTGAATCTTCTGAAGATTGTCAATCTTCTAGACCca

ggacacgtaaatcactattgGGTCTAGAAGATTGA

atagtgatttacgtgtcc

After the LCR assembly, we isolated the CDS using these two primers(with BioBrick Prefix in the fwd primer and Suffix in the rev primer):

fwd:GAATTCGCGGCCGCTTCTAGatgGGTTCTACTCAAGttt

rev:TACTAGTAGCGGCCGCTGCAGAGATTGACAATCTTCAGAAGAT

References

[1]Zhou H, Zha Z, Liu Y, et al. Structural Insights into the Down-regulation of Overexpressed p185her2/neu Protein of Transformed Cells by the Antibody chA21[J]. Journal of Biological Chemistry, 2011, 286(36): 31676-31683.

@@ Line 12: / Line 12: @@
 This coding sequence was synthesized by LCR assmebly using 36 oilgos
-AATTCCGattgtgtcc	                     R0
+AATTCCGattgtgtcc
-ggacacaatCGGAATTCATGGGTTCTACTCAAG	     F0
-CATATCAGTACCAGTACAAACTTGAGTAGAACCCATG	     R16
+ggacacaatCGGAATTCATGGGTTCTACTCAAG
-TTTGTACTGGTACTGATATGAAGTTGAGATTGCCAG	     F33
-AATGAGTTTCTGGAGAAGCTGGCAATCTCAACTT	     R53
+CATATCAGTACCAGTACAAACTTGAGTAGAACCCATG
-CTTCTCCAGAAACTCATTTGGATATGTTGAGACATTT	     F69
-CTTGACAACCTTGGTACAAATGTCTCAACATATCCA	     R87
+TTTGTACTGGTACTGATATGAAGTTGAGATTGCCAG
-GTACCAAGGTTGTCAAGTTGTTCAAGGTAACTTGG	     F106
-TGGCAAGTAAGTCAATTCCAAGTTACCTTGAACAA	     R123
+AATGAGTTTCTGGAGAAGCTGGCAATCTCAACTT
-AATTGACTTACTTGCCAACTAACGCTTCTTTGTC	     F141
-TCTTGAATATCTTGCAAAAAAGACAAAGAAGCGTTAGT	     R158
+CTTCTCCAGAAACTCATTTGGATATGTTGAGACATTT
-TTTTTTGCAAGATATTCAAGAAGTTCAAGGTTACGTTT	     F175
-CTTGGTTATGAGCAATCAAAACGTAACCTTGAACT	     R196
+CTTGACAACCTTGGTACAAATGTCTCAACATATCCA
-TGATTGCTCATAACCAAGTTAGACAAGTTCCATTGC	     F213
-CTCTAACAATTCTCAATCTTTGCAATGGAACTTGTCTAA	     R231
+GTACCAAGGTTGTCAAGTTGTTCAAGGTAACTTGG
-AAAGATTGAGAATTGTTAGAGGTACTCAATTGTTTGAAGAT    F249
-CAGCCAAAGCGTAGTTATCTTCAAACAATTGAGTAC	     R270
+TGGCAAGTAAGTCAATTCCAAGTTACCTTGAACAA
-AACTACGCTTTGGCTGTTTTGGATAACGGTGATC	     F290
-GGAGTAGTGTTGTTCAATGGATCACCGTTATCCAAAA	     R306
+AATTGACTTACTTGCCAACTAACGCTTCTTTGTC
-CATTGAACAACACTACTCCAGTTACTGGTGCTTCT	     F324
-CTCTCAAACCACCTGGAGAAGCACCAGTAACT	     R343
+TCTTGAATATCTTGCAAAAAAGACAAAGAAGCGTTAGT
-CCAGGTGGTTTGAGAGAATTGCAATTGAGATCTTT	     F359
-CCCTTCAAAATTTCAGTCAAAGATCTCAATTGCAATT	     R375
+TTTTTTGCAAGATATTCAAGAAGTTCAAGGTTACGTTT
-GACTGAAATTTTGAAGGGTGGTGTTTTGATTCAAAG	     F394
-ACACAATTGTGGGTTTCTTTGAATCAAAACACCA	     R412
+CTTGGTTATGAGCAATCAAAACGTAACCTTGAACT
-AAACCCACAATTGTGTTACCAAGATACTATTTTGTGG	     F430
-TGTTCTTATGAAAAATATCCTTCCACAAAATAGTATCTTGGTA  R446
+TGATTGCTCATAACCAAGTTAGACAAGTTCCATTGC
-AAGGATATTTTTCATAAGAACAACCAATTGGCTTTGAC	     F467
-GATCTGTTAGTATCAATCAAAGTCAAAGCCAATTGGT	     R489
+CTCTAACAATTCTCAATCTTTGCAATGGAACTTGTCTAA
-TTTGATTGATACTAACAGATCCAGAGCTTGTCATCCA	     F505
-CTTACACATTGGAGAACATGGATGACAAGCTCTG	     R526
+AAAGATTGAGAATTGTTAGAGGTACTCAATTGTTTGAAGAT
-TGTTCTCCAATGTGTAAGGGTTCCAGATGTTGGG	     F542
-CAATCTTCAGAAGATTCACCCCAACATCTGGAACC	     R560
+CAGCCAAAGCGTAGTTATCTTCAAACAATTGAGTAC
-GTGAATCTTCTGAAGATTGTCAATCTTCTAGACCca	     F576
-ggacacgtaaatcactattgGGTCTAGAAGATTGA	     R595
+AACTACGCTTTGGCTGTTTTGGATAACGGTGATC
-atagtgatttacgtgtcc	                     R612
+GGAGTAGTGTTGTTCAATGGATCACCGTTATCCAAAA
+CATTGAACAACACTACTCCAGTTACTGGTGCTTCT
+CTCTCAAACCACCTGGAGAAGCACCAGTAACT
+CCAGGTGGTTTGAGAGAATTGCAATTGAGATCTTT
+CCCTTCAAAATTTCAGTCAAAGATCTCAATTGCAATT
+GACTGAAATTTTGAAGGGTGGTGTTTTGATTCAAAG
+ACACAATTGTGGGTTTCTTTGAATCAAAACACCA
+AAACCCACAATTGTGTTACCAAGATACTATTTTGTGG
+TGTTCTTATGAAAAATATCCTTCCACAAAATAGTATCTTGGTA
+AAGGATATTTTTCATAAGAACAACCAATTGGCTTTGAC
+GATCTGTTAGTATCAATCAAAGTCAAAGCCAATTGGT
+TTTGATTGATACTAACAGATCCAGAGCTTGTCATCCA
+CTTACACATTGGAGAACATGGATGACAAGCTCTG
+TGTTCTCCAATGTGTAAGGGTTCCAGATGTTGGG
+CAATCTTCAGAAGATTCACCCCAACATCTGGAACC
+GTGAATCTTCTGAAGATTGTCAATCTTCTAGACCca
+ggacacgtaaatcactattgGGTCTAGAAGATTGA
+atagtgatttacgtgtcc
 ----
 After the LCR assembly, we isolated the CDS using these two primers(with BioBrick Prefix in the fwd primer and Suffix in the rev primer):
 fwd:GAATTCGCGGCCGCTTCTAGatgGGTTCTACTCAAGttt
 rev:TACTAGTAGCGGCCGCTGCAGAGATTGACAATCTTCAGAAGAT
 ===References===
 [1]Zhou H, Zha Z, Liu Y, et al. Structural Insights into the Down-regulation of Overexpressed p185her2/neu Protein of Transformed Cells by the Antibody chA21[J]. Journal of Biological Chemistry, 2011, 286(36): 31676-31683.