Difference between revisions of "Part:BBa K5490032:Design"

 
 
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===Design Notes===
 
===Design Notes===
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NONE
+
CasRx Part Design
 +
 
 +
Three inserts were ordered from the supplier for the CasRx constructs:
 +
CasRx1: This insert contains the first half of the effector protein with a Kozak sequence for efficient mammalian expression.
 +
CasRx2: This insert includes the second half of the effector protein, fused with an HA tag at the C-terminal end.
 +
CasRx3: The third insert incorporates an IRES sequence and an mCherry reporter, also tagged with a nuclear localization signal (NLS) at the C-terminal end.
 +
 
 +
The inserts were designed to have overlapping sequences with each other as well as with the borders of the vector after the removal of the luciferase gene. The vector pNFAT-RE-Luc10, provided by professor Meško and her team, contains a minimal promoter that exhibits high specificity for a synthetic NFAT transcription factor and the luciferase gene downstream. Two restriction enzymes, PspXI (upstream) and FseI (downstream), were identified at the gene borders. Additionally, it was ensured that after cleavage, the vector would have overlapping sequences with CasRx1 and CasRx3 inserts.
  
  

Latest revision as of 00:46, 29 September 2024


Responsive to synthetic NFAT CasRx


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1335
    Illegal EcoRI site found at 1755
    Illegal PstI site found at 4754
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1335
    Illegal EcoRI site found at 1755
    Illegal PstI site found at 4754
    Illegal NotI site found at 141
    Illegal NotI site found at 211
    Illegal NotI site found at 506
    Illegal NotI site found at 576
    Illegal NotI site found at 3744
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1335
    Illegal EcoRI site found at 1755
    Illegal BamHI site found at 3735
    Illegal BamHI site found at 3809
    Illegal XhoI site found at 806
    Illegal XhoI site found at 3210
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1335
    Illegal EcoRI site found at 1755
    Illegal PstI site found at 4754
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1335
    Illegal EcoRI site found at 1755
    Illegal PstI site found at 4754
    Illegal NgoMIV site found at 1023
    Illegal AgeI site found at 7
    Illegal AgeI site found at 347
    Illegal AgeI site found at 372
    Illegal AgeI site found at 712
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

CasRx Part Design

Three inserts were ordered from the supplier for the CasRx constructs: CasRx1: This insert contains the first half of the effector protein with a Kozak sequence for efficient mammalian expression. CasRx2: This insert includes the second half of the effector protein, fused with an HA tag at the C-terminal end. CasRx3: The third insert incorporates an IRES sequence and an mCherry reporter, also tagged with a nuclear localization signal (NLS) at the C-terminal end.

The inserts were designed to have overlapping sequences with each other as well as with the borders of the vector after the removal of the luciferase gene. The vector pNFAT-RE-Luc10, provided by professor Meško and her team, contains a minimal promoter that exhibits high specificity for a synthetic NFAT transcription factor and the luciferase gene downstream. Two restriction enzymes, PspXI (upstream) and FseI (downstream), were identified at the gene borders. Additionally, it was ensured that after cleavage, the vector would have overlapping sequences with CasRx1 and CasRx3 inserts.


Source

Composite part designed by IGEM IOANNINA 2024

References