Why does it appear: Before an SLIC/Gibson/CPEC/SLiCE or Golden Gate assembly is designed, j5 checks whether or not the Eugene design rules are satisfied for the construct to be assembled. If one or more rules are violated, the assembly design is aborted and the error message may provide additional information.

Why does it appear: j5 requires a sequence region bordering or containing assembly junction junction_number that is at least OVERHANGOVERLAPBPS bps in length (e.g. 4 bp) that will be exactly the same in all resulting assembled sequences in order to select an overhang/overlap sequence. If no such constant sequence region exists, then it will not be possible to design a scar-less SLIC/Gibson/CPEC/SLiCE or Golden Gate combinatorial assembly.

How to address it: Your options to address this include limiting the parts that will be included in the assembly of the combinatorial library, until a constant sequence region is achieved, or by purposely introducing a constant sequence region (introducing scars in the process) by adding constant bps (or making constant point mutations) to the ends of the assembly pieces (5'_piece and 3'_piece) at assembly junction junction_number.

Why does it appear: You have selected to force the overhang positions for assembly junctions junction_number and junction_number_2 as part of a SLIC/Gibson/CPEC/SLiCE or Golden Gate assembly process, but the resulting overhang/overlap sequences (overhang_overlap_sequence, and overhang_overlap_sequence_2) are incompatible, meaning that they will base pair with each other to an extent greater than that which is tolerable (by default, more than 2 out of the 4 bp are self-complementary for Golden Gate; this parameter is adjustable in the j5 parameters file). The error_message may provide additional information.

Why does it appear: You have selected to force the overhang/overlap position for assembly junction junction_number as part of a SLIC/Gibson/CPEC/SLiCE or Golden Gate assembly process, but the resulting overhang/overlap sequence (overhang_overlap_sequence) is self-incompatible, meaning that it will base pair with itself to an extent greater than that which is tolerable (by default, more than 2 out of the 4 bp are self-complementary for Golden Gate; this parameter is adjustable in the j5 parameters file). The error_message may provide additional information.

Why does it appear: You have selected to force the overhang position for assembly junction junction_number as part of a Golden Gate assembly process, but the resulting overhang sequence (overhang_sequence) is incompatible with an overhang resulting from the cleavage of a Golden Gate type IIs endonuclease site internal to one or more assembly pieces, meaning that it will base pair with the internal overhang to an extent greater than that which is tolerable (by default, more than 2 out of the 4 bp are self-complementary for Golden Gate; this parameter is adjustable in the j5 parameters file). The error_message may provide additional information.

Why does it appear: In order to minimize the number of oligos required for a combinatorial SLIC/Gibson/CPEC/SLiCE or Golden Gate assembly (i.e. to maximally re-use PCR products and minimize the number of PCR products required), j5 requires that overhangs/overlaps are located about the assembly junctions such that all base-pairs that the overhangs/overlaps span are absolutely conserved across all desired combinations. This error will only occur when a forced relative overhang position is specified for a junction. For more information, refer to our brief survey of j5 functionality documentation, concerning "when j5 designs a combinatorial Golden Gate assembly, the overhang search space about a given combinatorial assembly junction is constrained".

Why does it appear: j5 exhaustively searches through all possible combinations of available overhangs/overlaps for the assembly junctions. Occasionally (usually only if there are many junctions and the sequence regions around the junctions from which to select overhangs/overlaps are highly constrained), it is possible that j5 can not find a set of overhangs/overlaps that are compatible with themselves and with each other.

What to do when you receive this error: If you receive this error, it will be necessary to introduce additional scar base pairs at one or more assembly junctions, to increase the number and diversity of the overhangs/overlaps from which to select.

Why does it appear: When digest linearizing the vector backbone (which precludes the possibility of adding any flanking sequence), the vector backbone sequence itself must be at least a Gibson overlap long (26 bp is the j5 default; if you set an alternative length using the j5 parameters input file, GIBSONOVERLAPBPS will reflect your chosen length). It seems unlikely that one would ever digest linearize a vector backbone that isn't at least a Gibson overlap long, but j5 checks just in case.

What does it mean: The extra (5' or 3') CPEC overlap bps setting specified in the target part order list file for assembly piece assembly_piece is forcing j5 to design an overlap sequence that is shorter than the minimum overlap length specified in the j5 parameters file.

Why does it appear: This can occur if the (5' or 3') CPEC overlap bps setting for assembly piece assembly_piece is too negative, and there isn't enough space on the other side of the overlap in which to expand (for example, because of a degenerate base pair or the end of sequence identity among all combinatorial variants), or the other side of the overlap is also fixed by a (3' or 5') CPEC overlap bps setting for the (subsequent or previous) assembly piece.

What does it mean: The extra (5' or 3') CPEC overlap bps setting specified in the target part order list file for assembly piece assembly_piece is forcing j5 to design an overlap sequence that extends beyond the allowable region for the overlap (which can be constrained by a degenerate base pair or the end of sequence identity among all combinatorial variants).

Why does it appear: This can occur if the (5' or 3') CPEC overlap bps setting for assembly piece assembly_piece is too positive. There isn't enough space in which to expand, or the other side of the overlap is fixed by a negative (3' or 5') CPEC overlap bps setting for the (subsequent or previous) assembly piece.