Difference between revisions of "Part:BBa K5490036"
 
Line 19: Line 19:
 
<partinfo>BBa_K5490036 parameters</partinfo>
 
<partinfo>BBa_K5490036 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
BUILDING CAS13D IN 10REPMIN FLUC
 +
 +
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
 +
 +
 
Meško M, Lebar T, Dekleva P, Jerala R, Benčina M. Engineering and Rewiring of a Calcium-Dependent Signaling Pathway. ACS Synth Biol. 2020 Aug 21;9(8):2055-2065. doi: 10.1021/acssynbio.0c00133. Epub 2020 Jul 20. PMID: 32643923; PMCID: PMC7467823.
 
Meško M, Lebar T, Dekleva P, Jerala R, Benčina M. Engineering and Rewiring of a Calcium-Dependent Signaling Pathway. ACS Synth Biol. 2020 Aug 21;9(8):2055-2065. doi: 10.1021/acssynbio.0c00133. Epub 2020 Jul 20. PMID: 32643923; PMCID: PMC7467823.

Latest revision as of 23:40, 28 September 2024


10x[AB]-pMIN-fL

Is a type of promoter with high specificity to a particular type of transcription factor, in this case, the synthetic NFAT (BBa_K5490017), it achieves this by having various upstream elements of a small DNA sequence that contains the TATA box where polymerase II will bind. "Downstream, it contains a luciferase gene for assessing promoter strength through a luciferase assay.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1270
    Illegal EcoRI site found at 5571
    Illegal XbaI site found at 1457
    Illegal XbaI site found at 3239
    Illegal SpeI site found at 5325
    Illegal PstI site found at 1451
    Illegal PstI site found at 4414
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1270
    Illegal EcoRI site found at 5571
    Illegal SpeI site found at 5325
    Illegal PstI site found at 1451
    Illegal PstI site found at 4414
    Illegal NotI site found at 4393
    Illegal NotI site found at 5717
    Illegal NotI site found at 5787
    Illegal NotI site found at 6082
    Illegal NotI site found at 6152
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1270
    Illegal EcoRI site found at 5571
    Illegal BamHI site found at 1719
    Illegal XhoI site found at 6339
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1270
    Illegal EcoRI site found at 5571
    Illegal XbaI site found at 1457
    Illegal XbaI site found at 3239
    Illegal SpeI site found at 5325
    Illegal PstI site found at 1451
    Illegal PstI site found at 4414
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1270
    Illegal EcoRI site found at 5571
    Illegal XbaI site found at 1457
    Illegal XbaI site found at 3239
    Illegal SpeI site found at 5325
    Illegal PstI site found at 1451
    Illegal PstI site found at 4414
    Illegal NgoMIV site found at 1028
    Illegal NgoMIV site found at 1049
    Illegal NgoMIV site found at 1364
    Illegal NgoMIV site found at 1472
    Illegal NgoMIV site found at 2950
    Illegal NgoMIV site found at 2967
    Illegal NgoMIV site found at 3099
    Illegal AgeI site found at 752
    Illegal AgeI site found at 3245
    Illegal AgeI site found at 5583
    Illegal AgeI site found at 5923
    Illegal AgeI site found at 5948
    Illegal AgeI site found at 6288
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1740
    Illegal SapI site found at 3451
    Illegal SapI.rc site found at 934


BUILDING CAS13D IN 10REPMIN FLUC

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


Meško M, Lebar T, Dekleva P, Jerala R, Benčina M. Engineering and Rewiring of a Calcium-Dependent Signaling Pathway. ACS Synth Biol. 2020 Aug 21;9(8):2055-2065. doi: 10.1021/acssynbio.0c00133. Epub 2020 Jul 20. PMID: 32643923; PMCID: PMC7467823.