Difference between revisions of "Part:pSB1K04"

 
m
Line 4: Line 4:
  
 
pOdd4 Loop Vector based on pSB1K3
 
pOdd4 Loop Vector based on pSB1K3
 +
 +
 +
===iGEM TypeIIS Vectors===
 +
{{TypeIIS/Plasmids}}
 +
 +
 +
{{TypeIIS/References}}
 +
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 16:33, 15 June 2020


pOdd4 Loop Vector based on pSB1K3

pOdd4 Loop Vector based on pSB1K3


iGEM TypeIIS Vectors

Universal Acceptor Vector (for Level 0)


pOdd Loop Vectors (for Level 1)

Assembling into Level 1 Plasmds

pSB1K01-pSB1K04 series of plasmids are based on the pOdd set of Loop Vectors and are built upon the pSB1K3 plasmid backbone.

Note:

  • Since pSB1K3 is a high-copy plasmid backbone, these vectors should not be used for Level 3 assemblies.
  • Currently, Registry samples of these plasmid backbones have not been fully sequenced. Only their prefix and suffix along with their insert BBa_J04454 have been verified.


TypeIIS-Level1-J04454-Simple.png
Simplified diagram of BBa_J04454 and pSB1K0# with BsaI cut site.


When four Level 0 parts are assembled into the plasmid backbone (paired with BBa_J04454) in a one-pot assembly with BsaI...

TypeIIS-Level1-L0-Part-Assembly-Simple.png


the resulting transcriptional unit (TU) will be flanked by SapI restriction sites and the specified 3 bp Fusion Sites (NNN).

TypeIIS-Level1-TU-Simple.png



Level 1 Plasmid Set

Level 1 transcriptional units in this set of plasmid backbones will be flanked by SapI recognition sites and specified 3bp fusion sites, enabling further assembly of up to 4 transcriptional units as a Level 2 Assembly.

Registry Name Loop Name Fusion Site 5' TU Fusion Site 3'
pSB1K01 pOdd1 ATG TU 1 GCA
pSB1K02 pOdd2 GCA TU 2 TAC
pSB1K03 pOdd3 TAC TU 3 CAG
pSB1K04 pOdd4 CAG TU 4 GGT

Level 1 -> Level 2 Assembly

Up to 4 Level 1 transcriptional units can be assembled into a Level 2 multi-transcriptional unit. By planning your assemb

TypeIIS-Level1-Plasmid-Set.png
Simplified diagram of pSB1K0# set with assembled transcriptional units (TUs).


pEven Loop Vectors (for Level 2)

When four Level 1 parts (TUs) are assembled into a Multi-Transcriptional Unit (MTU) into the following plasmid backbones (with BBa_J04455), the MTU will be flanked by BsaI restriction sites and these 4 bp Fusion Sites.

Registry Name Loop Name Fusion Site 5' MTU Fusion Site 3'
pSB3C01 pEven1 GGAG MTU 1 TACT
pSB3C02 pEven2 TACT MTU 2 AATG
pSB3C03 pEven3 AATG MTU 3 GCTT
pSB3C04 pEven4 GCTT MTU 4 CGCT

Note:

  • The documented sequence shows an additional BsaI site. This would impact assembly out of (but not into) this set of vectors. A new set of vectors should be created or used in the long term.
  • Currently, Registry samples of these plasmid backbones have not been fully sequenced. Only their prefix and suffix along with their insert BBa_J04455 have been verified.


The iGEM Type IIS assembly standard is based on MoClo and Loop


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2179
    Illegal PstI site found at 12
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2179
    Illegal PstI site found at 12
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2179
    Illegal XhoI site found at 1029
    Illegal XhoI site found at 2055
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2179
    Illegal PstI site found at 12
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2179
    Illegal PstI site found at 12
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 2185
    Illegal SapI.rc site found at 5