pydna 5.5.4__py3-none-any.whl → 5.5.5__py3-none-any.whl

pydna/assembly2.py

@@ -4,29 +4,29 @@ Improved implementation of the assembly module. To see a list of issues with the
 see [issues tagged with fixed-with-new-assembly-model](https://github.com/pydna-group/pydna/issues?q=is%3Aissue%20state%3Aopen%20label%3Afixed-with-new-assembly-model)
 """
 
-import networkx as _nx
-import itertools as _itertools
+import networkx as nx
+import itertools
 from Bio.SeqFeature import SimpleLocation, Location
-from Bio.Seq import reverse_complement
+
 from Bio.Restriction.Restriction import RestrictionBatch
 import regex
 import copy
 
 from pydna.utils import (
-    shift_location as _shift_location,
+    shift_location,
     flatten,
-    location_boundaries as _location_boundaries,
-    locations_overlap as _locations_overlap,
+    location_boundaries,
+    locations_overlap,
     sum_is_sticky,
     limit_iterator,
     create_location,
 )
-from pydna._pretty import pretty_str as _pretty_str
+from pydna._pretty import pretty_str as ps
 from pydna.common_sub_strings import common_sub_strings as common_sub_strings_str
-from pydna.dseqrecord import Dseqrecord as _Dseqrecord
-from pydna.dseq import Dseq as _Dseq
-from pydna.primer import Primer as _Primer
-from pydna.seqrecord import SeqRecord as _SeqRecord
+from pydna.dseqrecord import Dseqrecord
+from pydna.dseq import Dseq
+from pydna.primer import Primer
+from pydna.seqrecord import SeqRecord
 from pydna.types import (
     CutSiteType,
     # TODO: allow user to enforce multi-site
@@ -38,6 +38,7 @@ from pydna.types import (
 )
 from pydna.gateway import gateway_overlap, find_gateway_sites
 from pydna.cre_lox import cre_loxP_overlap
+from pydna.alphabet import anneal_strands
 
 from typing import TYPE_CHECKING, Callable, Literal
 from pydna.opencloning_models import (
@@ -59,7 +60,7 @@ from pydna.crispr import cas9
 import warnings
 
 if TYPE_CHECKING:  # pragma: no cover
-    from Bio.Restriction import AbstractCut as _AbstractCut
+    from Bio.Restriction import AbstractCut
 
 
 def gather_overlapping_locations(
@@ -71,29 +72,29 @@ def gather_overlapping_locations(
     the output will be [(loc1, loc2), (loc3,)].
     """
     # Make a graph with all the locations as nodes
-    G = _nx.Graph()
+    G = nx.Graph()
     for i, loc in enumerate(locs):
         G.add_node(i, location=loc)
 
     # Add edges between nodes that overlap
     for i in range(len(locs)):
         for j in range(i + 1, len(locs)):
-            if _locations_overlap(locs[i], locs[j], fragment_length):
+            if locations_overlap(locs[i], locs[j], fragment_length):
                 G.add_edge(i, j)
 
     # Get groups of overlapping locations
     groups = list()
-    for loc_set in _nx.connected_components(G):
+    for loc_set in nx.connected_components(G):
         groups.append(tuple(locs[i] for i in loc_set))
 
     # Sort by location of the first element in each group (does not matter which since they are overlapping)
-    groups.sort(key=lambda x: _location_boundaries(x[0])[0])
+    groups.sort(key=lambda x: location_boundaries(x[0])[0])
 
     return groups
 
 
 def ends_from_cutsite(
-    cutsite: CutSiteType, seq: _Dseq
+    cutsite: CutSiteType, seq: Dseq
 ) -> tuple[tuple[str, str], tuple[str, str]]:
     """Get the sticky or blunt ends created by a restriction enzyme cut.
 
@@ -116,7 +117,7 @@ def ends_from_cutsite(
         and the sequence of the overhang. The first tuple is for the left end, second for the right end.
 
     >>> from Bio.Restriction import NotI
-    >>> x = _Dseq("ctcgGCGGCCGCcagcggccg")
+    >>> x = Dseq("ctcgGCGGCCGCcagcggccg")
     >>> x.get_cutsites(NotI)
     [((6, -4), NotI)]
     >>> ends_from_cutsite(x.get_cutsites(NotI)[0], x)
@@ -143,8 +144,8 @@ def ends_from_cutsite(
 
 
 def restriction_ligation_overlap(
-    seqx: _Dseqrecord,
-    seqy: _Dseqrecord,
+    seqx: Dseqrecord,
+    seqy: Dseqrecord,
     enzymes=RestrictionBatch,
     partial=False,
     allow_blunt=False,
@@ -155,9 +156,9 @@ def restriction_ligation_overlap(
 
     Parameters
     ----------
-    seqx : _Dseqrecord
+    seqx : Dseqrecord
         The first sequence
-    seqy : _Dseqrecord
+    seqy : Dseqrecord
         The second sequence
     enzymes : RestrictionBatch
         The enzymes to use
@@ -211,7 +212,7 @@ def restriction_ligation_overlap(
     #     if not seqy.circular:
     #         cuts_y.append(((0, 0), None))
     matches = list()
-    for cut_x, cut_y in _itertools.product(cuts_x, cuts_y):
+    for cut_x, cut_y in itertools.product(cuts_x, cuts_y):
         # A blunt end
         if allow_blunt and cut_x[0][1] == cut_y[0][1] == 0:
             matches.append((cut_x[0][0], cut_y[0][0], 0))
@@ -255,7 +256,7 @@ def combine_algorithms(*algorithms: AssemblyAlgorithmType) -> AssemblyAlgorithmT
 
 
 def blunt_overlap(
-    seqx: _Dseqrecord, seqy: _Dseqrecord, limit=None
+    seqx: Dseqrecord, seqy: Dseqrecord, limit=None
 ) -> list[SequenceOverlap]:
     """
     Assembly algorithm to find blunt overlaps. Used for blunt ligation.
@@ -265,9 +266,9 @@ def blunt_overlap(
 
     Parameters
     ----------
-    seqx : _Dseqrecord
+    seqx : Dseqrecord
         The first sequence
-    seqy : _Dseqrecord
+    seqy : Dseqrecord
         The second sequence
     limit : int
         There for compatibility, but it is ignored
@@ -293,7 +294,7 @@ def blunt_overlap(
 
 
 def common_sub_strings(
-    seqx: _Dseqrecord, seqy: _Dseqrecord, limit=25
+    seqx: Dseqrecord, seqy: Dseqrecord, limit=25
 ) -> list[SequenceOverlap]:
     """
     Assembly algorithm to find common substrings of length == limit. see the docs of
@@ -356,7 +357,7 @@ def common_sub_strings(
     return [r for r in results if r not in shifted_matches]
 
 
-def gibson_overlap(seqx: _Dseqrecord, seqy: _Dseqrecord, limit=25):
+def gibson_overlap(seqx: Dseqrecord, seqy: Dseqrecord, limit=25):
     """
     Assembly algorithm to find terminal overlaps (e.g. for Gibson assembly).
     The order matters, we want alignments like:
@@ -375,9 +376,9 @@ def gibson_overlap(seqx: _Dseqrecord, seqy: _Dseqrecord, limit=25):
 
     Parameters
     ----------
-    seqx : _Dseqrecord
+    seqx : Dseqrecord
         The first sequence
-    seqy : _Dseqrecord
+    seqy : Dseqrecord
         The second sequence
     limit : int
         Minimum length of the overlap
@@ -402,9 +403,9 @@ def gibson_overlap(seqx: _Dseqrecord, seqy: _Dseqrecord, limit=25):
     # This is only relevant for linear fragments, so we don't need to worry about
     # shifting locations for circular fragments.
     trim_x_left = -seqx.seq.ovhg if seqx.seq.ovhg < 0 else 0
-    trim_x_right = seqx.seq.watson_ovhg() if seqx.seq.watson_ovhg() < 0 else None
+    trim_x_right = seqx.seq.watson_ovhg if seqx.seq.watson_ovhg < 0 else None
     trim_y_left = -seqy.seq.ovhg if seqy.seq.ovhg < 0 else 0
-    trim_y_right = seqy.seq.watson_ovhg() if seqy.seq.watson_ovhg() < 0 else None
+    trim_y_right = seqy.seq.watson_ovhg if seqy.seq.watson_ovhg < 0 else None
 
     stringx = str(seqx.seq[trim_x_left:trim_x_right]).upper()
     stringy = str(seqy.seq[trim_y_left:trim_y_right]).upper()
@@ -422,7 +423,7 @@ def gibson_overlap(seqx: _Dseqrecord, seqy: _Dseqrecord, limit=25):
     return [tuple(m) for m in matches]
 
 
-def sticky_end_sub_strings(seqx: _Dseqrecord, seqy: _Dseqrecord, limit: bool = False):
+def sticky_end_sub_strings(seqx: Dseqrecord, seqy: Dseqrecord, limit: bool = False):
     """
     Assembly algorithm for ligation of sticky ends.
 
@@ -431,9 +432,9 @@ def sticky_end_sub_strings(seqx: _Dseqrecord, seqy: _Dseqrecord, limit: bool = F
 
     Parameters
     ----------
-    seqx : _Dseqrecord
+    seqx : Dseqrecord
         The first sequence
-    seqy : _Dseqrecord
+    seqy : Dseqrecord
         The second sequence
     limit : bool
         Whether to allow partial overlaps
@@ -466,6 +467,7 @@ def sticky_end_sub_strings(seqx: _Dseqrecord, seqy: _Dseqrecord, limit: bool = F
     [(4, 0, 2)]
 
     """
+
     overlap = sum_is_sticky(
         seqx.seq.three_prime_end(), seqy.seq.five_prime_end(), limit
     )
@@ -475,7 +477,7 @@ def sticky_end_sub_strings(seqx: _Dseqrecord, seqy: _Dseqrecord, limit: bool = F
 
 
 def zip_match_leftwards(
-    seqx: _SeqRecord, seqy: _SeqRecord, match: SequenceOverlap
+    seqx: SeqRecord, seqy: SeqRecord, match: SequenceOverlap
 ) -> SequenceOverlap:
     """
     Starting from the rightmost edge of the match, return a new match encompassing the max
@@ -483,15 +485,15 @@ def zip_match_leftwards(
     than the limit or a shorter match if there are mismatches. This is convenient to maintain
     as many features as possible. It is used in PCR assembly.
 
-    >>> seq = _Dseqrecord('AAAAACGTCCCGT')
-    >>> primer = _Dseqrecord('ACGTCCCGT')
+    >>> seq = Dseqrecord('AAAAACGTCCCGT')
+    >>> primer = Dseqrecord('ACGTCCCGT')
     >>> match = (13, 9, 0) # an empty match at the end of each
     >>> zip_match_leftwards(seq, primer, match)
     (4, 0, 9)
 
     Works in circular molecules if the match spans the origin:
-    >>> seq = _Dseqrecord('TCCCGTAAAAACG', circular=True)
-    >>> primer = _Dseqrecord('ACGTCCCGT')
+    >>> seq = Dseqrecord('TCCCGTAAAAACG', circular=True)
+    >>> primer = Dseqrecord('ACGTCCCGT')
     >>> match = (6, 9, 0)
     >>> zip_match_leftwards(seq, primer, match)
     (10, 0, 9)
@@ -512,11 +514,11 @@ def zip_match_leftwards(
     # For those cases we shift by length, then go back
 
     end_on_x = match[0] + match[2]
-    if isinstance(seqx, _Dseqrecord) and seqx.circular and end_on_x <= len(seqx):
+    if isinstance(seqx, Dseqrecord) and seqx.circular and end_on_x <= len(seqx):
         end_on_x += len(seqx)
 
     end_on_y = match[1] + match[2]
-    if isinstance(seqy, _Dseqrecord) and seqy.circular and end_on_y <= len(seqy):
+    if isinstance(seqy, Dseqrecord) and seqy.circular and end_on_y <= len(seqy):
         end_on_y += len(seqy)
 
     count = 0
@@ -533,7 +535,7 @@ def zip_match_leftwards(
 
 
 def zip_match_rightwards(
-    seqx: _Dseqrecord, seqy: _Dseqrecord, match: SequenceOverlap
+    seqx: Dseqrecord, seqy: Dseqrecord, match: SequenceOverlap
 ) -> SequenceOverlap:
     """Same as zip_match_leftwards, but towards the right."""
 
@@ -549,19 +551,19 @@ def zip_match_rightwards(
     return (start_on_x, start_on_y, count)
 
 
-def seqrecord2_uppercase_DNA_string(seqr: _SeqRecord) -> str:
+def seqrecord2_uppercase_DNA_string(seqr: SeqRecord) -> str:
     """
     Transform a Dseqrecord to a sequence string where U is replaced by T, everything is upper case and
     circular sequences are repeated twice. This is used for PCR, to support primers with U's (e.g. for USER cloning).
     """
     out = str(seqr.seq).upper().replace("U", "T")
-    if isinstance(seqr, _Dseqrecord) and seqr.circular:
+    if isinstance(seqr, Dseqrecord) and seqr.circular:
         return out * 2
     return out
 
 
 def primer_template_overlap(
-    seqx: _Dseqrecord | _Primer, seqy: _Dseqrecord | _Primer, limit=25, mismatches=0
+    seqx: Dseqrecord | Primer, seqy: Dseqrecord | Primer, limit=25, mismatches=0
 ) -> list[SequenceOverlap]:
     """
     Assembly algorithm to find overlaps between a primer and a template. It accepts mismatches.
@@ -573,9 +575,9 @@ def primer_template_overlap(
 
     Parameters
     ----------
-    seqx : _Dseqrecord | _Primer
+    seqx : Dseqrecord | Primer
         The primer
-    seqy : _Dseqrecord | _Primer
+    seqy : Dseqrecord | Primer
         The template
     limit : int
         Minimum length of the overlap
@@ -604,11 +606,11 @@ def primer_template_overlap(
     []
     """
 
-    if isinstance(seqx, _Primer) and isinstance(seqy, _Dseqrecord):
+    if isinstance(seqx, Primer) and isinstance(seqy, Dseqrecord):
         primer = seqx
         template = seqy
         reverse_primer = False
-    elif isinstance(seqx, _Dseqrecord) and isinstance(seqy, _Primer):
+    elif isinstance(seqx, Dseqrecord) and isinstance(seqy, Primer):
         primer = seqy
         template = seqx
         reverse_primer = True
@@ -662,45 +664,8 @@ def primer_template_overlap(
     return list(sorted(out))
 
 
-def fill_left(seq: _Dseq) -> _Dseq:
-    """Fill the left overhang of a sequence with the complementary sequence."""
-    new_watson = seq.watson
-    new_crick = seq.crick
-
-    # Watson 5' overhang
-    if seq.ovhg < 0:
-        new_crick = new_crick + reverse_complement(seq.watson[: -seq.ovhg])
-    # Crick 5' overhang
-    elif seq.ovhg > 0:
-        new_watson = reverse_complement(seq.crick[-seq.ovhg :]) + new_watson
-
-    return _Dseq(new_watson, new_crick, 0)
-
-
-def fill_right(seq: _Dseq) -> _Dseq:
-    """Fill the right overhang of a sequence with the complementary sequence."""
-    new_watson = seq.watson
-    new_crick = seq.crick
-
-    # Watson 3' overhang
-    watson_ovhg = seq.watson_ovhg()
-    if watson_ovhg < 0:
-        new_watson = new_watson + reverse_complement(seq.crick[:-watson_ovhg])
-
-    # Crick 3' overhang
-    elif watson_ovhg > 0:
-        new_crick = reverse_complement(seq.watson[-watson_ovhg:]) + new_crick
-
-    return _Dseq(new_watson, new_crick, seq.ovhg)
-
-
-def fill_dseq(seq: _Dseq) -> _Dseq:
-    """Fill the overhangs of a sequence with the complementary sequence."""
-    return fill_left(fill_right(seq))
-
-
 def reverse_complement_assembly(
-    assembly: EdgeRepresentationAssembly, fragments: list[_Dseqrecord]
+    assembly: EdgeRepresentationAssembly, fragments: list[Dseqrecord]
 ) -> EdgeRepresentationAssembly:
     """Complement an assembly, i.e. reverse the order of the fragments and the orientation of the overlaps."""
     new_assembly = list()
@@ -714,7 +679,7 @@ def reverse_complement_assembly(
 def filter_linear_subassemblies(
     linear_assemblies: list[EdgeRepresentationAssembly],
     circular_assemblies: list[EdgeRepresentationAssembly],
-    fragments: list[_Dseqrecord],
+    fragments: list[Dseqrecord],
 ) -> list[EdgeRepresentationAssembly]:
     """Remove linear assemblies which are sub-assemblies of circular assemblies"""
     all_circular_assemblies = circular_assemblies + [
@@ -773,7 +738,7 @@ def assembly2str_tuple(assembly: EdgeRepresentationAssembly) -> str:
 
 
 def assembly_has_mismatches(
-    fragments: list[_Dseqrecord], assembly: EdgeRepresentationAssembly
+    fragments: list[Dseqrecord], assembly: EdgeRepresentationAssembly
 ) -> bool:
     """Check if an assembly has mismatches. This should never happen and if so it returns an error."""
     for u, v, loc_u, loc_v in assembly:
@@ -789,7 +754,7 @@ def assembly_has_mismatches(
 
 
 def assembly_is_circular(
-    assembly: EdgeRepresentationAssembly, fragments: list[_Dseqrecord]
+    assembly: EdgeRepresentationAssembly, fragments: list[Dseqrecord]
 ) -> bool:
     """
     Based on the topology of the locations of an assembly, determine if it is circular.
@@ -798,22 +763,22 @@ def assembly_is_circular(
     if assembly[0][0] != assembly[-1][1]:
         return False
     elif (
-        isinstance(fragments[abs(assembly[0][0]) - 1], _Dseqrecord)
+        isinstance(fragments[abs(assembly[0][0]) - 1], Dseqrecord)
         and fragments[abs(assembly[0][0]) - 1].circular
     ):
         return True
     else:
         return (
-            _location_boundaries(assembly[0][2])[0]
-            > _location_boundaries(assembly[-1][3])[0]
+            location_boundaries(assembly[0][2])[0]
+            > location_boundaries(assembly[-1][3])[0]
         )
 
 
 def assemble(
-    fragments: list[_Dseqrecord],
+    fragments: list[Dseqrecord],
     assembly: EdgeRepresentationAssembly,
     is_insertion: bool = False,
-) -> _Dseqrecord:
+) -> Dseqrecord:
     """Generate a Dseqrecord from an assembly and a list of fragments."""
 
     if is_insertion:
@@ -830,14 +795,15 @@ def assemble(
         u, v, loc_u, loc_v = asm_edge
         f_u = fragments[u - 1] if u > 0 else fragments[-u - 1].reverse_complement()
         f_v = fragments[v - 1] if v > 0 else fragments[-v - 1].reverse_complement()
-        seq_u = str(loc_u.extract(f_u).seq).upper()
-        seq_v = str(loc_v.extract(f_v).seq).upper()
-        if seq_u != seq_v:
+        seq_u = str(loc_u.extract(f_u).seq)
+        seq_v = str(loc_v.extract(f_v).seq.rc())
+        # Test if seq_u and seq_v anneal
+        if not anneal_strands(seq_u, seq_v):
             raise ValueError("Mismatch in assembly")
 
     # We transform into Dseqrecords (for primers)
     dseqr_fragments = [
-        f if isinstance(f, _Dseqrecord) else _Dseqrecord(f) for f in fragments
+        f if isinstance(f, Dseqrecord) else Dseqrecord(f) for f in fragments
     ]
     subfragments = get_assembly_subfragments(
         dseqr_fragments, subfragment_representation
@@ -845,42 +811,23 @@ def assemble(
 
     # Length of the overlaps between consecutive assembly fragments
     fragment_overlaps = [len(e[-1]) for e in assembly]
+    out_dseqrecord = subfragments.pop(0)
 
-    out_dseqrecord = _Dseqrecord(subfragments[0])
+    for fragment, overlap in zip(subfragments, fragment_overlaps):
+        out_dseqrecord.seq = out_dseqrecord.seq.cast_to_ds_right()
+        out_dseqrecord.seq = out_dseqrecord.seq.exo1_end(overlap)
+        fragment.seq = fragment.seq.cast_to_ds_left()
+        fragment.seq = fragment.seq.exo1_front(overlap)
+        out_dseqrecord += fragment
 
-    for fragment, overlap in zip(subfragments[1:], fragment_overlaps):
-        # Shift the features of the right fragment to the left by ``overlap``
-        new_features = [
-            f._shift(len(out_dseqrecord) - overlap) for f in fragment.features
-        ]
-        # Join the left sequence including the overlap with the right sequence without the overlap
-        # we use fill_right / fill_left so that it works for ligation of sticky ends
-        out_dseqrecord = _Dseqrecord(
-            fill_right(out_dseqrecord.seq) + fill_left(fragment.seq)[overlap:],
-            features=out_dseqrecord.features + new_features,
-        )
-
-    # For circular assemblies, close the loop and wrap origin-spanning features
+    # For circular assemblies, process the fragment and loop
     if is_circular:
+        out_dseqrecord.seq = out_dseqrecord.seq.cast_to_ds_left()
+        out_dseqrecord.seq = out_dseqrecord.seq.cast_to_ds_right()
         overlap = fragment_overlaps[-1]
-
-        # Special case for blunt circularisation
-        if overlap == 0:
-            out_dseqrecord = out_dseqrecord.looped()
-        else:
-            # Remove trailing overlap
-            out_dseqrecord = _Dseqrecord(
-                fill_dseq(out_dseqrecord.seq)[:-overlap],
-                features=out_dseqrecord.features,
-                circular=True,
-            )
-            for feature in out_dseqrecord.features:
-                start, end = _location_boundaries(feature.location)
-                if start >= len(out_dseqrecord) or end > len(out_dseqrecord):
-                    # Wrap around the origin
-                    feature.location = _shift_location(
-                        feature.location, 0, len(out_dseqrecord)
-                    )
+        out_dseqrecord.seq = out_dseqrecord.seq.exo1_front(overlap)
+        out_dseqrecord.seq = out_dseqrecord.seq.exo1_end(overlap)
+        out_dseqrecord = out_dseqrecord.looped()
 
     out_dseqrecord.source = AssemblySource.from_subfragment_representation(
         subfragment_representation, fragments, is_circular
@@ -889,8 +836,8 @@ def assemble(
 
 
 def annotate_primer_binding_sites(
-    input_dseqr: _Dseqrecord, fragments: list[_Dseqrecord]
-) -> _Dseqrecord:
+    input_dseqr: Dseqrecord, fragments: list[Dseqrecord]
+) -> Dseqrecord:
     """Annotate the primer binding sites in a Dseqrecord."""
     fwd, _, rvs = fragments
     start_rvs = len(input_dseqr) - len(rvs)
@@ -970,9 +917,9 @@ def subfragment_representation2edge_representation(
 
 
 def get_assembly_subfragments(
-    fragments: list[_Dseqrecord],
+    fragments: list[Dseqrecord],
     subfragment_representation: SubFragmentRepresentationAssembly,
-) -> list[_Dseqrecord]:
+) -> list[Dseqrecord]:
     """From the fragment representation returned by edge_representation2subfragment_representation, get the subfragments that are joined together.
 
     Subfragments are the slices of the fragments that are joined together
@@ -1013,19 +960,26 @@ def get_assembly_subfragments(
 
 
 def extract_subfragment(
-    seq: _Dseqrecord, start_location: Location, end_location: Location
-) -> _Dseqrecord:
+    seq: Dseqrecord, start_location: Location | None, end_location: Location | None
+) -> Dseqrecord:
     """Extract a subfragment from a sequence for an assembly, given the start and end locations of the subfragment."""
-    start = 0 if start_location is None else _location_boundaries(start_location)[0]
-    end = None if end_location is None else _location_boundaries(end_location)[1]
+
+    if seq.circular and (start_location is None or end_location is None):
+        raise ValueError(
+            "Start and end locations cannot be None for circular sequences"
+        )
+        # This could be used to have consistent behaviour for circular sequences, where the start is arbitrary. However,
+        # they should never get None, so this is not used.
+        # if start_location is None:
+        #     start_location = end_location
+        # elif end_location is None:
+        #     end_location = start_location
+
+    start = 0 if start_location is None else location_boundaries(start_location)[0]
+    end = None if end_location is None else location_boundaries(end_location)[1]
 
     # Special case, some of it could be handled by better Dseqrecord slicing in the future
-    if (
-        seq.circular
-        and start_location is not None
-        and end_location is not None
-        and _locations_overlap(start_location, end_location, len(seq))
-    ):
+    if seq.circular and locations_overlap(start_location, end_location, len(seq)):
         # The overhang is different for origin-spanning features, for instance
         # for a feature join{[12:13], [0:3]} in a sequence of length 13, the overhang
         # is -4, not 9
@@ -1035,7 +989,7 @@ def extract_subfragment(
             ovhg = 0
         dummy_cut = ((start, ovhg), None)
         open_seq = seq.apply_cut(dummy_cut, dummy_cut)
-        return _Dseqrecord(fill_dseq(open_seq.seq), features=open_seq.features)
+        return Dseqrecord(open_seq.seq.cast_to_ds(), features=open_seq.features)
 
     return seq[start:end]
 
@@ -1178,14 +1132,15 @@ class Assembly:
 
     def __init__(
         self,
-        frags: list[_Dseqrecord],
+        frags: list[Dseqrecord],
         limit: int = 25,
         algorithm: AssemblyAlgorithmType = common_sub_strings,
         use_fragment_order: bool = True,
         use_all_fragments: bool = False,
     ):
+
         # TODO: allow for the same fragment to be included more than once?
-        self.G = _nx.MultiDiGraph()
+        self.G = nx.MultiDiGraph()
         # Add positive and negative nodes for forward and reverse fragments
         self.G.add_nodes_from((i + 1, {"seq": f}) for (i, f) in enumerate(frags))
         self.G.add_nodes_from(
@@ -1193,12 +1148,12 @@ class Assembly:
         )
 
         # Iterate over all possible combinations of fragments
-        fragment_pairs = _itertools.combinations(
+        fragment_pairs = itertools.combinations(
             filter(lambda x: x > 0, self.G.nodes), 2
         )
         for i, j in fragment_pairs:
             # All the relative orientations of the fragments in the pair
-            for u, v in _itertools.product([i, -i], [j, -j]):
+            for u, v in itertools.product([i, -i], [j, -j]):
                 u_seq = self.G.nodes[u]["seq"]
                 v_seq = self.G.nodes[v]["seq"]
                 matches = algorithm(u_seq, v_seq, limit)
@@ -1216,7 +1171,7 @@ class Assembly:
     @classmethod
     def assembly_is_valid(
         cls,
-        fragments: list[_Dseqrecord | _Primer],
+        fragments: list[Dseqrecord | Primer],
         assembly: EdgeRepresentationAssembly,
         is_circular: bool,
         use_all_fragments: bool,
@@ -1232,6 +1187,23 @@ class Assembly:
         if len(assembly) == 0:
             return False
 
+        # Topology check -> Circular sequences cannot be first or last in a linear assembly.
+        # For example, let's imagine aACGTc (linear) and gACGTc (circular).
+        # It should not be possible to join them into a linear assembly. It's similar if we
+        # think of a restriction-ligation assembly, example: aGAATTCc (linear) and gGAATTCc
+        # (circular).
+        # A linear product can be generated where the circular molecule is cut open, and one end
+        # it joins the linear molecule and on the other it's free, but for now it's not a
+        # relevant product and it's excluded.
+        first_fragment = fragments[abs(assembly[0][0]) - 1]
+        last_fragment = fragments[abs(assembly[-1][1]) - 1]
+        if not is_circular and (
+            isinstance(first_fragment, Dseqrecord)
+            and first_fragment.circular
+            or (isinstance(last_fragment, Dseqrecord) and last_fragment.circular)
+        ):
+            return False
+
         if use_all_fragments and len(fragments) != len(
             set(flatten(map(abs, e[:2]) for e in assembly))
         ):
@@ -1269,8 +1241,8 @@ class Assembly:
             # Incompatible as described in figure above
             fragment = fragments[abs(v1) - 1]
             if (
-                isinstance(fragment, _Primer) or not fragment.circular
-            ) and _location_boundaries(start_location)[1] >= _location_boundaries(
+                isinstance(fragment, Primer) or not fragment.circular
+            ) and location_boundaries(start_location)[1] >= location_boundaries(
                 end_location
             )[
                 1
@@ -1294,8 +1266,8 @@ class Assembly:
         match: SequenceOverlap,
         u: int,
         v: int,
-        first: _Dseqrecord,
-        secnd: _Dseqrecord,
+        first: Dseqrecord,
+        secnd: Dseqrecord,
     ):
         """Add edges to the graph from a match returned by the ``algorithm`` function (see pydna.common_substrings). For
         format of edges (see documentation of the Assembly class).
@@ -1314,10 +1286,10 @@ class Assembly:
         else:
             # We use shift_location with 0 to wrap origin-spanning features
             locs = [
-                _shift_location(
+                shift_location(
                     SimpleLocation(x_start, x_start + length), 0, len(first)
                 ),
-                _shift_location(
+                shift_location(
                     SimpleLocation(y_start, y_start + length), 0, len(secnd)
                 ),
             ]
@@ -1352,7 +1324,7 @@ class Assembly:
         """
 
         # Copy the graph since we will add the begin and end mock nodes
-        G = _nx.MultiDiGraph(self.G)
+        G = nx.MultiDiGraph(self.G)
         G.add_nodes_from(["begin", "end"])
 
         if self.use_fragment_order:
@@ -1390,7 +1362,7 @@ class Assembly:
     def node_path2assembly_list(
         self, cycle: list[int], circular: bool
     ) -> list[EdgeRepresentationAssembly]:
-        """Convert a node path in the format [1, 2, 3] (as returned by _nx.cycles.simple_cycles) to a list of all
+        """Convert a node path in the format [1, 2, 3] (as returned by networkx.cycles.simple_cycles) to a list of all
           possible assemblies.
 
         There may be multiple assemblies for a given node path, if there are several edges connecting two nodes,
@@ -1404,11 +1376,11 @@ class Assembly:
             combine.append([(u, v, key) for key in self.G[u][v]])
         return [
             tuple(map(self.format_assembly_edge, x))
-            for x in _itertools.product(*combine)
+            for x in itertools.product(*combine)
         ]
 
     def get_unique_linear_paths(
-        self, G_with_begin_end: _nx.MultiDiGraph, max_paths=10000
+        self, G_with_begin_end: nx.MultiDiGraph, max_paths=10000
     ) -> list[list[int]]:
         """Get unique linear paths from the graph, removing those that contain the same node twice."""
         # We remove the begin and end nodes, and get all paths without edges
@@ -1419,8 +1391,8 @@ class Assembly:
         node_paths = [
             x[1:-1]
             for x in limit_iterator(
-                _nx.all_simple_paths(
-                    _nx.DiGraph(G_with_begin_end),
+                nx.all_simple_paths(
+                    nx.DiGraph(G_with_begin_end),
                     "begin",
                     "end",
                     cutoff=(len(self.fragments) + 1),
@@ -1469,7 +1441,7 @@ class Assembly:
         sorted_cycles = map(
             circular_permutation_min_abs,
             limit_iterator(
-                _nx.cycles.simple_cycles(self.G, length_bound=len(self.fragments)),
+                nx.cycles.simple_cycles(self.G, length_bound=len(self.fragments)),
                 10000,
             ),
         )
@@ -1534,8 +1506,8 @@ class Assembly:
             fragment = self.fragments[abs(v1) - 1]
             # Find the pair of edges that should be last and first  ((3, 1, [8:10], [9:11)]), (1, 2, [4:6], [0:2]) in
             # the example above. Only one of the pairs of edges should satisfy this condition for the topology to make sense.
-            left_of_insertion = _location_boundaries(start_location)[0]
-            right_of_insertion = _location_boundaries(end_location)[0]
+            left_of_insertion = location_boundaries(start_location)[0]
+            right_of_insertion = location_boundaries(end_location)[0]
             if not fragment.circular and (
                 right_of_insertion >= left_of_insertion
                 # The below condition is for single-site integration.
@@ -1547,7 +1519,7 @@ class Assembly:
                 #
                 # The locations of homology on the genome are [0:10] and [2:12], so not identical
                 # but they overlap.
-                or _locations_overlap(start_location, end_location, len(fragment))
+                or locations_overlap(start_location, end_location, len(fragment))
             ):
                 edge_pair_index.append(i)
 
@@ -1578,13 +1550,13 @@ class Assembly:
         fragment1 = self.fragments[abs(f1) - 1]
         fragment2 = self.fragments[abs(f2) - 1]
 
-        if not _locations_overlap(
+        if not locations_overlap(
             loc_f1_1, loc_f1_2, len(fragment1)
-        ) or not _locations_overlap(loc_f2_2, loc_f2_1, len(fragment2)):
+        ) or not locations_overlap(loc_f2_2, loc_f2_1, len(fragment2)):
             return same_assembly
 
         # Sort to make compatible with insertion assembly
-        if _location_boundaries(loc_f1_1)[0] > _location_boundaries(loc_f1_2)[0]:
+        if location_boundaries(loc_f1_1)[0] > location_boundaries(loc_f1_2)[0]:
             new_assembly = same_assembly[::-1]
         else:
             new_assembly = same_assembly[:]
@@ -1597,10 +1569,10 @@ class Assembly:
         fragment2 = self.fragments[abs(f2) - 1]
 
         # Extract boundaries
-        f2_1_start, _ = _location_boundaries(loc_f2_1)
-        f2_2_start, f2_2_end = _location_boundaries(loc_f2_2)
-        f1_1_start, _ = _location_boundaries(loc_f1_1)
-        f1_2_start, f1_2_end = _location_boundaries(loc_f1_2)
+        f2_1_start, _ = location_boundaries(loc_f2_1)
+        f2_2_start, f2_2_end = location_boundaries(loc_f2_2)
+        f1_1_start, _ = location_boundaries(loc_f1_1)
+        f1_2_start, f1_2_end = location_boundaries(loc_f1_2)
 
         overlap_diff = len(fragment1[f1_1_start:f1_2_end]) - len(
             fragment2[f2_1_start:f2_2_end]
@@ -1640,7 +1612,7 @@ class Assembly:
                 "only_adjacent_edges not implemented for insertion assemblies"
             )
 
-        cycles = limit_iterator(_nx.cycles.simple_cycles(self.G), 10000)
+        cycles = limit_iterator(nx.cycles.simple_cycles(self.G), 10000)
 
         # We apply constrains already here because sometimes the combinatorial explosion is too large
         if self.use_all_fragments:
@@ -1659,7 +1631,7 @@ class Assembly:
             )
 
         # We find cycles first
-        iterator = limit_iterator(_nx.cycles.simple_cycles(self.G), 10000)
+        iterator = limit_iterator(nx.cycles.simple_cycles(self.G), 10000)
         assemblies = sum(
             map(lambda x: self.node_path2assembly_list(x, True), iterator), []
         )
@@ -1683,21 +1655,19 @@ class Assembly:
 
     def assemble_linear(
         self, only_adjacent_edges: bool = False, max_assemblies: int = 50
-    ) -> list[_Dseqrecord]:
+    ) -> list[Dseqrecord]:
         """Assemble linear constructs, from assemblies returned by self.get_linear_assemblies."""
         assemblies = self.get_linear_assemblies(only_adjacent_edges, max_assemblies)
         return [assemble(self.fragments, a) for a in assemblies]
 
     def assemble_circular(
         self, only_adjacent_edges: bool = False, max_assemblies: int = 50
-    ) -> list[_Dseqrecord]:
+    ) -> list[Dseqrecord]:
         """Assemble circular constructs, from assemblies returned by self.get_circular_assemblies."""
         assemblies = self.get_circular_assemblies(only_adjacent_edges, max_assemblies)
         return [assemble(self.fragments, a) for a in assemblies]
 
-    def assemble_insertion(
-        self, only_adjacent_edges: bool = False
-    ) -> list[_Dseqrecord]:
+    def assemble_insertion(self, only_adjacent_edges: bool = False) -> list[Dseqrecord]:
         """Assemble insertion constructs, from assemblies returned by self.get_insertion_assemblies."""
         assemblies = self.get_insertion_assemblies(only_adjacent_edges)
         return [assemble(self.fragments, a, is_insertion=True) for a in assemblies]
@@ -1739,10 +1709,10 @@ class Assembly:
                         if edge_location not in this_dict[key]:
                             this_dict[key].append(edge_location)
             this_dict["left"] = sorted(
-                this_dict["left"], key=lambda x: _location_boundaries(x)[0]
+                this_dict["left"], key=lambda x: location_boundaries(x)[0]
             )
             this_dict["right"] = sorted(
-                this_dict["right"], key=lambda x: _location_boundaries(x)[0]
+                this_dict["right"], key=lambda x: location_boundaries(x)[0]
             )
             locations_on_fragments[node] = this_dict
 
@@ -1789,7 +1759,7 @@ class Assembly:
 
             pairs = list()
             for pair in zip(left, right):
-                pairs += list(_itertools.product(*pair))
+                pairs += list(itertools.product(*pair))
             allowed_location_pairs[node] = pairs
 
         fragment_assembly = edge_representation2subfragment_representation(
@@ -1802,7 +1772,7 @@ class Assembly:
 
     def __repr__(self):
         # https://pyformat.info
-        return _pretty_str(
+        return ps(
             "Assembly\n"
             "fragments..: {sequences}\n"
             "limit(bp)..: {limit}\n"
@@ -1823,7 +1793,7 @@ class PCRAssembly(Assembly):
     the number of mismatches allowed in the overlap. Only supports substitution mismatches, not indels.
     """
 
-    def __init__(self, frags: list[_Dseqrecord | _Primer], limit=25, mismatches=0):
+    def __init__(self, frags: list[Dseqrecord | Primer], limit=25, mismatches=0):
 
         value_error = ValueError(
             "PCRAssembly assembly must be initialised with a list/tuple of primer, template, primer"
@@ -1833,15 +1803,15 @@ class PCRAssembly(Assembly):
 
         # Validate the inputs: should be a series of primer, template, primer
         wrong_fragment_class = (
-            not isinstance(frags[0], _Primer),
-            isinstance(frags[1], _Primer),
-            not isinstance(frags[2], _Primer),
+            not isinstance(frags[0], Primer),
+            isinstance(frags[1], Primer),
+            not isinstance(frags[2], Primer),
         )
         if any(wrong_fragment_class):
             raise value_error
 
         # TODO: allow for the same fragment to be included more than once?
-        self.G = _nx.MultiDiGraph()
+        self.G = nx.MultiDiGraph()
         # Add positive and negative nodes for forward and reverse fragments
         self.G.add_nodes_from((i + 1, {"seq": f}) for (i, f) in enumerate(frags))
         self.G.add_nodes_from(
@@ -1854,8 +1824,8 @@ class PCRAssembly(Assembly):
             # primer, template, primer
             p1, t, p2 = (i + 1, i + 2, i + 3)
             primer_ids += [p1, p2]
-            pairs += list(_itertools.product([p1, p2], [t, -t]))
-            pairs += list(_itertools.product([t, -t], [-p1, -p2]))
+            pairs += list(itertools.product([p1, p2], [t, -t]))
+            pairs += list(itertools.product([t, -t], [-p1, -p2]))
 
         for u, v in pairs:
             u_seq = self.G.nodes[u]["seq"]
@@ -1894,20 +1864,33 @@ class PCRAssembly(Assembly):
             "get_insertion_assemblies not implemented for PCR assemblies"
         )
 
+    def assemble_linear(
+        self, only_adjacent_edges: bool = False, max_assemblies: int = 50
+    ) -> list[Dseqrecord]:
+        """
+        Overrides the parent method to ensure that the 5' of the crick strand of the product matches the
+        sequence of the reverse primer. This is important when using primers with dUTP (for USER cloning).
+        """
+        results = super().assemble_linear(only_adjacent_edges, max_assemblies)
+        for result in results:
+            rp = self.fragments[2]
+            result.seq = result.seq[: -len(rp)] + Dseq(str(rp.seq.rc()))
+        return results
+
 
 class SingleFragmentAssembly(Assembly):
     """
     An assembly that represents the circularisation or splicing of a single fragment.
     """
 
-    def __init__(self, frags: [_Dseqrecord], limit=25, algorithm=common_sub_strings):
+    def __init__(self, frags: [Dseqrecord], limit=25, algorithm=common_sub_strings):
 
         if len(frags) != 1:
             raise ValueError(
                 "SingleFragmentAssembly assembly must be initialised with a single fragment"
             )
         # TODO: allow for the same fragment to be included more than once?
-        self.G = _nx.MultiDiGraph()
+        self.G = nx.MultiDiGraph()
         frag = frags[0]
         # Add positive and negative nodes for forward and reverse fragments
         self.G.add_node(1, seq=frag)
@@ -1958,8 +1941,8 @@ class SingleFragmentAssembly(Assembly):
             if x[0][2] == x[0][3]:
                 return False
             # We don't want to get overlap only (e.g. GAATTCcatGAATTC giving GAATTC)
-            left_start, _ = _location_boundaries(x[0][2])
-            _, right_end = _location_boundaries(x[0][3])
+            left_start, _ = location_boundaries(x[0][2])
+            _, right_end = location_boundaries(x[0][3])
             if left_start == 0 and right_end == len(self.fragments[0]):
                 return False
             return True
@@ -1982,18 +1965,19 @@ class SingleFragmentAssembly(Assembly):
 
 
 def common_function_assembly_products(
-    frags: list[_Dseqrecord],
+    frags: list[Dseqrecord],
     limit: int | None,
     algorithm: Callable,
     circular_only: bool,
     filter_results_function: Callable | None = None,
-) -> list[_Dseqrecord]:
+    only_adjacent_edges: bool = False,
+) -> list[Dseqrecord]:
     """Common function to avoid code duplication. Could be simplified further
     once SingleFragmentAssembly and Assembly are merged.
 
     Parameters
     ----------
-    frags : list[_Dseqrecord]
+    frags : list[Dseqrecord]
         List of DNA fragments to assemble
     limit : int or None
         Minimum overlap length required, or None if not applicable
@@ -2001,10 +1985,14 @@ def common_function_assembly_products(
         Function that determines valid overlaps between fragments
     circular_only : bool
         If True, only return circular assemblies
+    filter_results_function : Callable or None
+        Function that filters the results
+    only_adjacent_edges : bool
+        If True, only return assemblies that use only adjacent edges
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List of assembled DNA molecules
     """
     if len(frags) == 1:
@@ -2013,10 +2001,10 @@ def common_function_assembly_products(
         asm = Assembly(
             frags, limit, algorithm, use_fragment_order=False, use_all_fragments=True
         )
-    output_assemblies = asm.get_circular_assemblies()
+    output_assemblies = asm.get_circular_assemblies(only_adjacent_edges)
     if not circular_only and len(frags) > 1:
         output_assemblies += filter_linear_subassemblies(
-            asm.get_linear_assemblies(), output_assemblies, frags
+            asm.get_linear_assemblies(only_adjacent_edges), output_assemblies, frags
         )
     if not circular_only and len(frags) == 1:
         output_assemblies += asm.get_insertion_assemblies()
@@ -2028,28 +2016,28 @@ def common_function_assembly_products(
 
 
 def _recast_sources(
-    products: list[_Dseqrecord], source_cls, **extra_fields
-) -> list[_Dseqrecord]:
+    products: list[Dseqrecord], source_cls, **extra_fields
+) -> list[Dseqrecord]:
     """Recast the `source` of each product to `source_cls` with optional extras.
 
     This avoids repeating the same for-loop across many assembly functions.
     """
     for prod in products:
         prod.source = source_cls(
-            **prod.source.model_dump(),
+            **prod.source.to_unserialized_dict(),
             **extra_fields,
         )
     return products
 
 
 def gibson_assembly(
-    frags: list[_Dseqrecord], limit: int = 25, circular_only: bool = False
-) -> list[_Dseqrecord]:
+    frags: list[Dseqrecord], limit: int = 25, circular_only: bool = False
+) -> list[Dseqrecord]:
     """Returns the products for Gibson assembly.
 
     Parameters
     ----------
-    frags : list[_Dseqrecord]
+    frags : list[Dseqrecord]
         List of DNA fragments to assemble
     limit : int, optional
         Minimum overlap length required, by default 25
@@ -2058,7 +2046,7 @@ def gibson_assembly(
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List of assembled DNA molecules
     """
 
@@ -2069,14 +2057,14 @@ def gibson_assembly(
 
 
 def in_fusion_assembly(
-    frags: list[_Dseqrecord], limit: int = 25, circular_only: bool = False
-) -> list[_Dseqrecord]:
+    frags: list[Dseqrecord], limit: int = 25, circular_only: bool = False
+) -> list[Dseqrecord]:
     """Returns the products for in-fusion assembly. This is the same as Gibson
     assembly, but with a different name.
 
     Parameters
     ----------
-    frags : list[_Dseqrecord]
+    frags : list[Dseqrecord]
         List of DNA fragments to assemble
     limit : int, optional
         Minimum overlap length required, by default 25
@@ -2085,7 +2073,7 @@ def in_fusion_assembly(
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List of assembled DNA molecules
     """
 
@@ -2094,14 +2082,14 @@ def in_fusion_assembly(
 
 
 def fusion_pcr_assembly(
-    frags: list[_Dseqrecord], limit: int = 25, circular_only: bool = False
-) -> list[_Dseqrecord]:
+    frags: list[Dseqrecord], limit: int = 25, circular_only: bool = False
+) -> list[Dseqrecord]:
     """Returns the products for fusion PCR assembly. This is the same as Gibson
     assembly, but with a different name.
 
     Parameters
     ----------
-    frags : list[_Dseqrecord]
+    frags : list[Dseqrecord]
         List of DNA fragments to assemble
     limit : int, optional
         Minimum overlap length required, by default 25
@@ -2110,7 +2098,7 @@ def fusion_pcr_assembly(
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List of assembled DNA molecules
     """
     products = gibson_assembly(frags, limit)
@@ -2118,13 +2106,13 @@ def fusion_pcr_assembly(
 
 
 def in_vivo_assembly(
-    frags: list[_Dseqrecord], limit: int = 25, circular_only: bool = False
-) -> list[_Dseqrecord]:
+    frags: list[Dseqrecord], limit: int = 25, circular_only: bool = False
+) -> list[Dseqrecord]:
     """Returns the products for in vivo assembly (IVA), which relies on homologous recombination between the fragments.
 
     Parameters
     ----------
-    frags : list[_Dseqrecord]
+    frags : list[Dseqrecord]
         List of DNA fragments to assemble
     limit : int, optional
         Minimum overlap length required, by default 25
@@ -2133,7 +2121,7 @@ def in_vivo_assembly(
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List of assembled DNA molecules
     """
     products = common_function_assembly_products(
@@ -2143,11 +2131,11 @@ def in_vivo_assembly(
 
 
 def restriction_ligation_assembly(
-    frags: list[_Dseqrecord],
-    enzymes: list["_AbstractCut"],
+    frags: list[Dseqrecord],
+    enzymes: list["AbstractCut"],
     allow_blunt: bool = True,
     circular_only: bool = False,
-) -> list[_Dseqrecord]:
+) -> list[Dseqrecord]:
     """Returns the products for restriction ligation assembly:
 
     - Finds cutsites in the fragments
@@ -2156,9 +2144,9 @@ def restriction_ligation_assembly(
 
     Parameters
     ----------
-    frags : list[_Dseqrecord]
+    frags : list[Dseqrecord]
         List of DNA fragments to assemble
-    enzymes : list[_AbstractCut]
+    enzymes : list[AbstractCut]
         List of restriction enzymes to use
     allow_blunt : bool, optional
         If True, allow blunt end ligations, by default True
@@ -2167,7 +2155,7 @@ def restriction_ligation_assembly(
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List of assembled DNA molecules
 
     Examples
@@ -2214,7 +2202,7 @@ def restriction_ligation_assembly(
         return restriction_ligation_overlap(x, y, enzymes, False, allow_blunt)
 
     products = common_function_assembly_products(
-        frags, None, algorithm_fn, circular_only
+        frags, None, algorithm_fn, circular_only, only_adjacent_edges=True
     )
     return _recast_sources(
         products, RestrictionAndLigationSource, restriction_enzymes=enzymes
@@ -2222,20 +2210,20 @@ def restriction_ligation_assembly(
 
 
 def golden_gate_assembly(
-    frags: list[_Dseqrecord],
-    enzymes: list["_AbstractCut"],
+    frags: list[Dseqrecord],
+    enzymes: list["AbstractCut"],
     allow_blunt: bool = True,
     circular_only: bool = False,
-) -> list[_Dseqrecord]:
+) -> list[Dseqrecord]:
     """Returns the products for Golden Gate assembly. This is the same as
     restriction ligation assembly, but with a different name. Check the documentation
     for ``restriction_ligation_assembly`` for more details.
 
     Parameters
     ----------
-    frags : list[_Dseqrecord]
+    frags : list[Dseqrecord]
         List of DNA fragments to assemble
-    enzymes : list[_AbstractCut]
+    enzymes : list[AbstractCut]
         List of restriction enzymes to use
     allow_blunt : bool, optional
         If True, allow blunt end ligations, by default True
@@ -2244,7 +2232,7 @@ def golden_gate_assembly(
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List of assembled DNA molecules
 
     Examples
@@ -2255,11 +2243,11 @@ def golden_gate_assembly(
 
 
 def ligation_assembly(
-    frags: list[_Dseqrecord],
+    frags: list[Dseqrecord],
     allow_blunt: bool = False,
     allow_partial_overlap: bool = False,
     circular_only: bool = False,
-) -> list[_Dseqrecord]:
+) -> list[Dseqrecord]:
     """Returns the products for ligation assembly, as inputs pass the fragments (digested if needed) that
     will be ligated.
 
@@ -2267,7 +2255,7 @@ def ligation_assembly(
 
     Parameters
     ----------
-    frags : list[_Dseqrecord]
+    frags : list[Dseqrecord]
         List of DNA fragments to assemble
     allow_blunt : bool, optional
         If True, allow blunt end ligations, by default False
@@ -2278,7 +2266,7 @@ def ligation_assembly(
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List of assembled DNA molecules
 
 
@@ -2333,17 +2321,17 @@ def assembly_is_multi_site(asm: list[EdgeRepresentationAssembly]) -> bool:
 
 
 def gateway_assembly(
-    frags: list[_Dseqrecord],
+    frags: list[Dseqrecord],
     reaction_type: Literal["BP", "LR"],
     greedy: bool = False,
     circular_only: bool = False,
     multi_site_only: bool = False,
-) -> list[_Dseqrecord]:
+) -> list[Dseqrecord]:
     """Returns the products for Gateway assembly / Gateway cloning.
 
     Parameters
     ----------
-    frags : list[_Dseqrecord]
+    frags : list[Dseqrecord]
         List of DNA fragments to assemble
     reaction_type : Literal['BP', 'LR']
         Type of Gateway reaction
@@ -2359,7 +2347,7 @@ def gateway_assembly(
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List of assembled DNA molecules
 
 
@@ -2446,13 +2434,13 @@ def gateway_assembly(
 
 
 def common_function_integration_products(
-    frags: list[_Dseqrecord], limit: int | None, algorithm: Callable
-) -> list[_Dseqrecord]:
+    frags: list[Dseqrecord], limit: int | None, algorithm: Callable
+) -> list[Dseqrecord]:
     """Common function to avoid code duplication for integration products.
 
     Parameters
     ----------
-    frags : list[_Dseqrecord]
+    frags : list[Dseqrecord]
         List of DNA fragments to integrate
     limit : int or None
         Minimum overlap length required, or None if not applicable
@@ -2461,7 +2449,7 @@ def common_function_integration_products(
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List of integrated DNA molecules
     """
     if len(frags) == 1:
@@ -2482,27 +2470,27 @@ def common_function_integration_products(
 
 
 def common_handle_insertion_fragments(
-    genome: _Dseqrecord, inserts: list[_Dseqrecord]
-) -> list[_Dseqrecord]:
+    genome: Dseqrecord, inserts: list[Dseqrecord]
+) -> list[Dseqrecord]:
     """Common function to handle / validate insertion fragments.
 
     Parameters
     ----------
-    genome : _Dseqrecord
+    genome : Dseqrecord
         Target genome sequence
-    inserts : list[_Dseqrecord] or _Dseqrecord
+    inserts : list[Dseqrecord] or Dseqrecord
         DNA fragment(s) to insert
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List containing genome and insert fragments
     """
-    if not isinstance(genome, _Dseqrecord):
+    if not isinstance(genome, Dseqrecord):
         raise ValueError("Genome must be a Dseqrecord object")
 
     if not isinstance(inserts, list) or not all(
-        isinstance(f, _Dseqrecord) for f in inserts
+        isinstance(f, Dseqrecord) for f in inserts
     ):
         raise ValueError("Inserts must be a list of Dseqrecord objects")
 
@@ -2513,13 +2501,13 @@ def common_handle_insertion_fragments(
 
 
 def common_function_excision_products(
-    genome: _Dseqrecord, limit: int | None, algorithm: Callable
-) -> list[_Dseqrecord]:
+    genome: Dseqrecord, limit: int | None, algorithm: Callable
+) -> list[Dseqrecord]:
     """Common function to avoid code duplication for excision products.
 
     Parameters
     ----------
-    genome : _Dseqrecord
+    genome : Dseqrecord
         Target genome sequence
     limit : int or None
         Minimum overlap length required, or None if not applicable
@@ -2528,7 +2516,7 @@ def common_function_excision_products(
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List of excised DNA molecules
     """
     asm = SingleFragmentAssembly([genome], limit, algorithm)
@@ -2536,25 +2524,25 @@ def common_function_excision_products(
 
 
 def homologous_recombination_integration(
-    genome: _Dseqrecord,
-    inserts: list[_Dseqrecord],
+    genome: Dseqrecord,
+    inserts: list[Dseqrecord],
     limit: int = 40,
-) -> list[_Dseqrecord]:
+) -> list[Dseqrecord]:
     """Returns the products resulting from the integration of an insert (or inserts joined
     through in vivo recombination) into the genome through homologous recombination.
 
     Parameters
     ----------
-    genome : _Dseqrecord
+    genome : Dseqrecord
         Target genome sequence
-    inserts : list[_Dseqrecord]
+    inserts : list[Dseqrecord]
         DNA fragment(s) to insert
     limit : int, optional
         Minimum homology length required, by default 40
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List of integrated DNA molecules
 
 
@@ -2590,21 +2578,21 @@ def homologous_recombination_integration(
 
 
 def homologous_recombination_excision(
-    genome: _Dseqrecord, limit: int = 40
-) -> list[_Dseqrecord]:
+    genome: Dseqrecord, limit: int = 40
+) -> list[Dseqrecord]:
     """Returns the products resulting from the excision of a fragment from the genome through
     homologous recombination.
 
     Parameters
     ----------
-    genome : _Dseqrecord
+    genome : Dseqrecord
         Target genome sequence
     limit : int, optional
         Minimum homology length required, by default 40
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List containing excised plasmid and remaining genome sequence
 
     Examples
@@ -2627,8 +2615,8 @@ def homologous_recombination_excision(
 
 
 def cre_lox_integration(
-    genome: _Dseqrecord, inserts: list[_Dseqrecord]
-) -> list[_Dseqrecord]:
+    genome: Dseqrecord, inserts: list[Dseqrecord]
+) -> list[Dseqrecord]:
     """Returns the products resulting from the integration of an insert (or inserts joined
     through cre-lox recombination among them) into the genome through cre-lox integration.
 
@@ -2636,14 +2624,14 @@ def cre_lox_integration(
 
     Parameters
     ----------
-    genome : _Dseqrecord
+    genome : Dseqrecord
         Target genome sequence
-    inserts : list[_Dseqrecord] or _Dseqrecord
+    inserts : list[Dseqrecord] or Dseqrecord
         DNA fragment(s) to insert
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List of integrated DNA molecules
 
     Examples
@@ -2686,17 +2674,17 @@ def cre_lox_integration(
     return _recast_sources(products, CreLoxRecombinationSource)
 
 
-def cre_lox_excision(genome: _Dseqrecord) -> list[_Dseqrecord]:
+def cre_lox_excision(genome: Dseqrecord) -> list[Dseqrecord]:
     """Returns the products for CRE-lox excision.
 
     Parameters
     ----------
-    genome : _Dseqrecord
+    genome : Dseqrecord
         Target genome sequence
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List containing excised plasmid and remaining genome sequence
 
     Examples
@@ -2738,28 +2726,28 @@ def cre_lox_excision(genome: _Dseqrecord) -> list[_Dseqrecord]:
 
 
 def crispr_integration(
-    genome: _Dseqrecord,
-    inserts: list[_Dseqrecord],
-    guides: list[_Primer],
+    genome: Dseqrecord,
+    inserts: list[Dseqrecord],
+    guides: list[Primer],
     limit: int = 40,
-) -> list[_Dseqrecord]:
+) -> list[Dseqrecord]:
     """
     Returns the products for CRISPR integration.
 
     Parameters
     ----------
-    genome : _Dseqrecord
+    genome : Dseqrecord
         Target genome sequence
-    inserts : list[_Dseqrecord]
+    inserts : list[Dseqrecord]
         DNA fragment(s) to insert
-    guides : list[_Primer]
+    guides : list[Primer]
         List of guide RNAs as Primer objects. This may change in the future.
     limit : int, optional
         Minimum overlap length required, by default 40
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List of integrated DNA molecules
 
     Examples
@@ -2804,8 +2792,9 @@ def crispr_integration(
     for i, product in enumerate(products):
         # The second element of product.source.input is conventionally the insert/repair fragment
         # The other two (first and third) are the two bits of the genome
-        repair_start = _location_boundaries(product.source.input[0].right_location)[0]
-        repair_end = _location_boundaries(product.source.input[2].left_location)[1]
+        repair_start = location_boundaries(product.source.input[0].right_location)[0]
+        # Here we do +1 because the position of the cut marks the boundary (e.g. 0:10, 10:20 if a cut is at pos 10)
+        repair_end = location_boundaries(product.source.input[2].left_location)[1] + 1
         repair_location = create_location(repair_start, repair_end, len(genome))
         some_cuts_inside_repair = []
         all_cuts_inside_repair = []
@@ -2836,22 +2825,22 @@ def crispr_integration(
 
 
 def pcr_assembly(
-    template: _Dseqrecord,
-    fwd_primer: _Primer,
-    rvs_primer: _Primer,
+    template: Dseqrecord,
+    fwd_primer: Primer,
+    rvs_primer: Primer,
     add_primer_features: bool = False,
     limit: int = 14,
     mismatches: int = 0,
-) -> list[_Dseqrecord]:
+) -> list[Dseqrecord]:
     """Returns the products for PCR assembly.
 
     Parameters
     ----------
-    template : _Dseqrecord
+    template : Dseqrecord
         Template sequence
-    fwd_primer : _Primer
+    fwd_primer : Primer
         Forward primer
-    rvs_primer : _Primer
+    rvs_primer : Primer
         Reverse primer
     add_primer_features : bool, optional
         If True, add primer features to the product, by default False
@@ -2862,7 +2851,7 @@ def pcr_assembly(
 
     Returns
     -------
-    list[_Dseqrecord]
+    list[Dseqrecord]
         List of assembled DNA molecules
     """