Part:pSB1K04

pOdd4 Loop Vector based on pSB1K3

pOdd4 Loop Vector based on pSB1K3

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.

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...

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

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

Simplified diagram of pSB1K0# set with assembled transcriptional units (TUs).

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

• A Modular Cloning System for Standardized Assembly of Multigene Constructs
• Loop assembly: a simple and open system for recursive fabrication of DNA circuits

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
  

Uppsala 2020's Addition

Since high copy number plasmids are not suitable for higher level assemblies, we have assembled 3 sets of new pOdd plasmids for different situations:

• pSB3K0# (BBa_K3425005 to BBa_K3425008): medium copy number (10-12 copies)
• pSB4K0# (BBa_K3425009 to BBa_K3425012): low copy number (~5 copies)
• pSB4A0# (BBa_K3425013 to BBa_K3425016): low copy number (~5 copies) with different resistance for cotransformations

TU_Dresden 2022's Addition

To make a plasmid suitable for yeast, we made several modification to pOdd vector, as well as added auxotrophy selective markers:

• URA3 marker BBa_K4365022
• HIS3 marker BBa_K4365024
• TRP1 marker BBa_K4365023