Adding constraints¶

Sequence constraints are central to Codeine’s design philosophy, allowing you to easily narrow the space of valid coding sequences to match your requirements.

Currently Codeine handles:

Forbidden motifs
Max homopolymer length
Restricted codon choices

As well as arbitrary combinations of the above. Let’s go through these!

Forbidden motifs¶

In biotechnology we often wish to exclude sequence motifs that interfere with cloning, synthesis, or downstream applications.

Codeine can exclude such sequences via forbidden_motifs:

from codeine import CodingSpace, RestrictionSite

aa_seq = 'MKTLEFQNGSCPRYKKL'

unconstrained = CodingSpace(aa_seq)

constrained = CodingSpace(
   aa_seq,
   forbidden_motifs=[
       'GAATTC',
       'CTCGAG',
   ],
   seed=42,
)

print(f'Without constraints: {unconstrained.n_valid_sequences:,} sequences')
print(f'With constraints: {constrained.n_valid_sequences:,} sequences')
print(constrained.sample())

For convenience, codeine.RestrictionSite provides a collection of commonly used restriction enzyme recognition sequences. For example:

from codeine import RestrictionSite

print(RestrictionSite.EcoRI)
print(RestrictionSite.BsaI)

This outputs a summary of each of these restriction sites:

EcoRI (GAATTC)
BsaI (GGTCTC / GAGACC)

The built-in motifs can then be used alongside custom motifs:

from codeine import CodingSpace, RestrictionSite

space = CodingSpace(
    aa_seq,
    forbidden_motifs=[
        RestrictionSite.EcoRI,
        RestrictionSite.BsaI,
        'GATTACA',
    ],
)

Each named restriction site is automatically interpreted to mean both the recognition sequence and its reverse complement.

The built-in restriction sites are:

BioBricks: EcoRI, XbaI, SpeI, PstI;
Common cloning: BamHI, HindIII, XhoI, SalI, KpnI, SacI, NcoI, NdeI, NotI, MluI, AgeI, AvrII, BglII;
Golden Gate: BsaI, BsmBI, BbsI, SapI.

The recognition sequences for these were taken from the New England Biolabs alphabetized list of recognition sequences.

Max homopolymer¶

Long homopolymer runs can be difficult to work with because they increase the risk of polymerase slippage and sequencing errors. In Codeine, runs of identical nucleotides can be avoided using the max_homopolymer constraint.

For example, setting max_homopolymer=5 excludes any coding sequence containing six or more consecutive identical nucleotides.

from codeine import CodingSpace

space = CodingSpace(
    aa_seq,
    max_homopolymer=5,
)

print(space.n_valid_sequences)

Fixed codons¶

codon_restrictions restricts which codons are allowed at specific amino acid positions. They can either be fixed exact codons, or subsets of the set of possible codons for that amino acid. Positions are 1-based.

from codeine import CodingSpace

aa_seq = 'SEQVENCE'

space = CodingSpace(
   aa_seq,
   codon_restrictions={
       1: ['TCG', 'TCA'],
       2: 'GAG',
   },
   seed=42,
)

Here, position 1 is restricted to TCG or TCA, and position 2 is fixed to GAG.

Combining constraints¶

Constraints can be combined freely!

from codeine import CodingSpace, RestrictionSite

aa_seq = 'MKTLEFQNGSCPRYKKL'

space = CodingSpace(
    aa_seq,
    codon_restrictions={
        2: ['AAA', 'AAG'],
        5: 'GAA',
    },
    forbidden_motifs=[
        RestrictionSite.EcoRI,
        RestrictionSite.XhoI,
        'TAGATA',
    ],
    max_homopolymer=5,
    seed=42,
)

print(space.n_valid_sequences)
print(space.sample())