Part:pSB3C03

pEven3 Loop Vector based on pSB3C5

pEven3 Loop Vector based on pSB3C5

iGEM Type IIS Vectors

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

• 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]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2713
  Illegal PstI site found at 12
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2713
  Illegal NheI site found at 1399
  Illegal PstI site found at 12
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2713
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2713
  Illegal PstI site found at 12
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2713
  Illegal PstI site found at 12
  Illegal AgeI site found at 990
  Illegal AgeI site found at 1313
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 285
  Illegal BsaI site found at 2719
  Illegal BsaI.rc site found at 6
  

Uppsala 2020's Improvement

TEAMNAME UofUppsala YEAR 2020 Authors Nuria Garriga Alonso, Bjorn Ancker Persson, Tereza Hubackova, Dorottya Marko, Hui Yu

The illegal BsaI site was removed by PCR mutagenesis to obtain the new pEven3 plasmid pSB3C13 (BBa_K3425003). This new plasmid was used for various clonings and it works as expected.

Moreover, pSB3C01 was also used for cloning and it also works as expected. However, we recommend using the new plasmids without an illegal BsaI site in order to avoid compromising the efficiency.