# Biobrick standard

This standard defines the required sequence properties for a Biobrick(tm) standard biological part. It does not define any functional characteristics of the parts, nor does it motivate any aspect of these standards. All sequences defined herein are specified in the 5' to 3' direction.

1. A Biobrick compatible standard biological part consists of a DNA fragment potentially conveying informational or functional properties to a composite structure assembled from multiple parts. The current assembly process requires certain sequence properties for the part and the surrounding DNA.
2. Allowed sequences within Biobrick parts include any DNA sequence which does not contain the following subsequences:
  EcoRI site:   GAATTC
XbaI site:    TCTAGA
SpeI site:    ACTAGT
PstI site:    CTGCAG
NotI site:    GCGGCCGC

1. Biobrick Suffix
• Each Biobrick part must contain precisely this sequence immediately following the 3' end of the part:
  T ACTAGT A GCGGCCG CTGCAG


1. Biobrick Prefix:

To allow the construction of ribosomal binding sequences 5' of coding regions, the prefix used for coding regions is distinguished from the sequence for non-coding Biobrick parts.

• For non-coding Biobrick parts (the default) the Biobrick part must contain precisely the following sequence immediately 5' of the part:
     GAATTC GCGGCCGC T TCTAGA G


• For Biobrick parts coding for proteins, the Biobrick part must contain precisely the following sequence immediately 5' of the ATG start of the coding region:
     GAATTC GCGGCCGC T TCTAG

• Parts containing start codons other than ATG must be modified to use ATG as the start codon. We highly recommend, but do not require, that all coding regions terminate with in-frame TAATAA stop codons, replacing other stop codons (TGA, TAG).

1. Plasmid context

Biobrick parts must be supplied in plasmids compatible with registry and assembly procedures. The pSBxxxx series of plasmids comply with these requirements. Other plasmids may be compliant with the following constraints:

• Antibiotic resistance: All plasmids must carry at least one of the following antibiotic resistance markers:
     Ampicillin
Chloramphenicol
Kanamycin
Tetracycline

• It is acceptable for a plasmid to convey both ampicillin resistance and no more than one additional antibiotic resistance markers.
• Note that certain strains convey resistance as well. Parts delivered to the registry must be in strains which do not convey resistance to these markers.

• Sequencing primers
• The registry uses the following primers in sequencing all parts. Plasmids which omit or misplace these primers cannot be sequenced:
     VF2:  TGCCACCTGACGTCTAAGAA
VR:   ATTACCGCCTTTGAGTGAGC


1. Strains
• The registry maintains parts libraries in frozen bacterial cultures, and encourages submissions in this form. The bacterial strain must be a K-12 cloning strain (endA-). Under no circumstances will the registry accept submissions in any strain which is not BSL-1. We recommend strains such as Top10, DH10B, and DH5a. We do not recommend submissions in MC4100, BL21 and similar strains.

Biobrick(tm) is a registered trademark of the Biobrick Foundation, Inc.