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logicgate1.png Synthetic Biology: Assembly Standards Guide


A distinguishing feature of synthetic biology is the ease with which parts can be combined to construct complex networks. This is accomplished through the use of assembly standards – combinations of restriction sites which preserve a standard prefix and suffix sequence at either end of the assembly, even as more parts are added. Therefore, the same reaction which ligates a promoter into an empty backbone can be repeated to ligate your gene of interest after the promoter, and so on.

Assembly_Standards_InfoGraphic_No_Scar.jpg

The following table provides information for the assembly standards most commonly used by part registries, including the specific prefix and suffix sequences for each (these can be built into PCR primers when creating a new part, with an additional 5' clamping sequence such as GTTTCTTC). Note that all prefix/suffix sequences are given for the coding strand, meaning that the suffix will need to be reverse complimented for primer design. The restriction enzymes used for assembly are the prohibited sites and must be omitted from the coding sequence of the part and backbone. For some standards, there are additional sites which should be avoided whenever possible.

Other popular cloning strategies for the assembly of synthetic parts include Gibson assembly and type IIS systems such as MoClo. For an overview of the most common cloning methods, click here.


BioBrick (RFC 10)

  • Prefix:
    GAATTC GCGGCCGC T TCTAGA G
  • Alternate Prefix:
    GAATTC GCGGCCGC T TCTAGA TG (contains start)
  • Prefix Enzymes:
    EcoR1, NotI, XbaI
  • Suffix:
    T ACTAGT A GCGGCCG CTGCAG
  • Suffix Enzymes:
    SpeI, NotI, PstI
  • Scar:
    TACTAGAG or TACTAG
  • Features:
    8 bp Scar; No In-Frame Fusions; Coding Prefix alleviates RBS-CDS issues.
  • Prohibited Restriction Sites:
    EcoRI, XbaI, SpeI, PstI
  • Restriction Sites to Avoid:
    NotI
  • For more info, visit iGEM:

BioBrick BB-2 (RFC 12)

  • Prefix:
    GAATTC GCGGCCGC T ACTAGT G
  • Prefix Enzymes:
    EcoR1, NotI, Spel
  • Suffix:
    GCTAGC GCGGCCG CTGCAG
  • Suffix Enzymes:
    Nhel, NotI, PstI
  • Scar:
    GCTAGT
  • Features:
    6 bp scar encodes Ala-Ser; Allows for in-frame fusions; NheI is rare in E. Coli genome.
  • Prohibited Restriction Sites:
    EcoRI, SpeI, NheI, PstI
  • Restriction Sites to Avoid:
    PvuII, XhoI, AvrII, XbaI, SapI
  • For more info, visit iGEM:

BglBrick – Berkeley Standard (RFC 21)

  • Prefix:
    GAATTC ATG AGATCT
  • Prefix Enzymes:
    EcoR1, BglII
  • Suffix:
    T GGATCC TAA CTCGAG
  • Suffix Enzymes:
    BamHI, Xhol
  • Scar:
    GGATCT
  • Features:
    Scar encodes Gly-Ser in-frame with prefix start codon; Employs high-efficiency enzymes.
  • Prohibited Restriction Sites:
    EcoRI, BglII, BamHI, XhoI
  • For more info, visit iGEM:

Silver Standard (RFC 23)

  • Prefix:
    GAATTC GCGGCCGC T TCTAGA G
  • Prefix Enzymes:
    EcoR1, NotI, XbaI
  • Suffix:
    T ACTAGT A GCGGCCG CTGCAG
  • Suffix Enzymes:
    SpeI, NotI, PstI
  • Scar:
    ACTAGA
  • Features:
    Modified RFC 10 with shortened prefix/suffix giving in-frame scar encoding Thr-Ala.
  • Prohibited Restriction Sites:
    EcoRI, XbaI, SpeI, PstI
  • For more info, visit iGEM:

Freiberg Standard (RFC 25)

  • Prefix:
    GAATTC GCGGCCGC T TCTAGA TG GCCGGC
  • Alternate Prefix:
    GAATTC GCGGCCGC T TCTAG (for "N-parts")
  • Prefix Enzymes:
    EcoR1, NotI, XbaI, (NgoMIV)
  • Suffix:
    ACCGGT TAAT ACTAGT A GCGGCCG CTGCAG
  • Suffix Enzymes:
    Agel, Spel, Notl, Pstl
  • Scar:
    ACCGGC
  • Features:
    Modified RFC 10 w/ start and stop codons; Creates in-frame fusions w/ linker Thr-Gly; Use N-parts prefix to preserve N-term sequence.
  • Prohibited Restriction Sites:
    EcoRI, XbaI, NgoMIV, AgeI, SpeI, PstI
  • For more info, visit iGEM: