opencloning 0.4.6__tar.gz → 0.4.7__tar.gz

{opencloning-0.4.6 → opencloning-0.4.7}/PKG-INFO

@@ -1,6 +1,6 @@
 Metadata-Version: 2.4
 Name: opencloning
-Version: 0.4.6
+Version: 0.4.7
 Summary: Backend of OpenCloning, a web application to generate molecular cloning strategies in json format, and share them with others.
 License: MIT
 License-File: LICENSE

{opencloning-0.4.6 → opencloning-0.4.7}/pyproject.toml

@@ -8,7 +8,7 @@ authors = ["Manuel Lera-Ramirez <manulera14@gmail.com>"]
 description = "Backend of OpenCloning, a web application to generate molecular cloning strategies in json format, and share them with others."
 license = "MIT"
 name = "opencloning"
-version = "0.4.6"
+version = "0.4.7"
 package-mode = true
 readme = "README.md"
 repository = "https://github.com/manulera/OpenCloning_backend"

{opencloning-0.4.6 → opencloning-0.4.7}/src/opencloning/ebic/primer_design.py

@@ -1,6 +1,6 @@
 from pydna.dseqrecord import Dseqrecord
 from Bio.SeqFeature import SimpleLocation
-from primer3 import bindings
+from ..primer3_functions import primer3_design_primers
 
 from ..pydantic_models import PrimerModel
 from .primer_design_settings import amanda_settings
@@ -13,23 +13,13 @@ adapter_right_fwd = 'ataGGTCTCtGCTT'
 adapter_right_rvs = 'ataGGTCTCtAGCG'
 
 
-def call_primer3(seq: str, seq_args: dict):
-    report = bindings.design_primers(
-        seq_args={
-            'SEQUENCE_ID': 'MH1000',
-            'SEQUENCE_TEMPLATE': seq,
-            **seq_args,
-        },
-        global_args=amanda_settings,
-    )
-    return report
-
-
 def ebic_primers(
     input_seq: Dseqrecord,
     location: SimpleLocation,
     max_inside: int,
     max_outside: int,
+    target_tm: float,
+    target_tm_tolerance: float,
 ) -> tuple[PrimerModel, PrimerModel, PrimerModel, PrimerModel]:
     """Design primers for EBIC"""
 
@@ -53,9 +43,13 @@ def ebic_primers(
         'SEQUENCE_PRIMER_PAIR_OK_REGION_LIST': f'0,{max_outside + max_inside},{len(right_template) - int(padding/2)},{int(padding/2)}',
     }
 
-    report_left = call_primer3(left_template, seq_args_left)
-    report_right = call_primer3(right_template, seq_args_right)
+    global_args = amanda_settings.copy()
+    global_args['PRIMER_OPT_TM'] = target_tm
+    global_args['PRIMER_MIN_TM'] = target_tm - target_tm_tolerance
+    global_args['PRIMER_MAX_TM'] = target_tm + target_tm_tolerance
 
+    report_left = primer3_design_primers(left_template, seq_args_left, global_args)
+    report_right = primer3_design_primers(right_template, seq_args_right, global_args)
     primer_names = ['left_fwd', 'left_rvs', 'right_fwd', 'right_rvs']
     primer_seqs = [
         adapter_left_fwd + report_left['PRIMER_LEFT'][0]['SEQUENCE'],

{opencloning-0.4.6 → opencloning-0.4.7}/src/opencloning/endpoints/primer_design.py

@@ -3,8 +3,6 @@ from pydantic import create_model
 import re
 from Bio.Restriction import RestrictionBatch
 from Bio.SeqUtils import gc_fraction
-from primer3 import bindings
-from itertools import product
 
 from ..dna_functions import get_invalid_enzyme_names
 from ..pydantic_models import PrimerModel, PrimerDesignQuery
@@ -14,6 +12,14 @@ from ..primer_design import (
     gibson_assembly_primers,
     simple_pair_primers,
 )
+from ..primer3_functions import (
+    primer3_calc_tm,
+    PrimerDesignSettings,
+    primer3_calc_homodimer,
+    primer3_calc_hairpin,
+    primer3_calc_heterodimer,
+    ThermodynamicResult,
+)
 from ..get_router import get_router
 from ..ebic.primer_design import ebic_primers
 from pydantic import BaseModel
@@ -45,6 +51,9 @@ def validate_spacers(spacers: list[str] | None, nb_templates: int, circular: boo
 async def primer_design_homologous_recombination(
     pcr_template: PrimerDesignQuery,
     homologous_recombination_target: PrimerDesignQuery,
+    settings: PrimerDesignSettings = Body(
+        ..., description='Primer design settings.', default_factory=PrimerDesignSettings
+    ),
     spacers: list[str] | None = Body(
         None,
         description='Spacers to add at the left and right side of the insertion.',
@@ -80,6 +89,7 @@ async def primer_design_homologous_recombination(
             insert_forward,
             target_tm,
             spacers,
+            tm_func=lambda x: primer3_calc_tm(x, settings),
         )
     except ValueError as e:
         raise HTTPException(400, *e.args)
@@ -93,6 +103,9 @@ async def primer_design_homologous_recombination(
 @router.post('/primer_design/gibson_assembly', response_model=PrimerDesignResponse)
 async def primer_design_gibson_assembly(
     pcr_templates: list[PrimerDesignQuery],
+    settings: PrimerDesignSettings = Body(
+        ..., description='Primer design settings.', default_factory=PrimerDesignSettings
+    ),
     spacers: list[str] | None = Body(
         None,
         description='Spacers to add between the restriction site and the 5\' end of the primer footprint (the part that binds the DNA).',
@@ -122,7 +135,13 @@ async def primer_design_gibson_assembly(
         templates.append(template)
     try:
         primers = gibson_assembly_primers(
-            templates, homology_length, minimal_hybridization_length, target_tm, circular, spacers
+            templates,
+            homology_length,
+            minimal_hybridization_length,
+            target_tm,
+            circular,
+            spacers,
+            tm_func=lambda x: primer3_calc_tm(x, settings),
         )
     except ValueError as e:
         raise HTTPException(400, *e.args)
@@ -137,6 +156,9 @@ async def primer_design_simple_pair(
         None,
         description='Spacers to add between the restriction site and the 5\' end of the primer footprint (the part that binds the DNA).',
     ),
+    settings: PrimerDesignSettings = Body(
+        ..., description='Primer design settings.', default_factory=PrimerDesignSettings
+    ),
     minimal_hybridization_length: int = Query(
         ..., description='The minimal length of the hybridization region in bps.'
     ),
@@ -186,6 +208,7 @@ async def primer_design_simple_pair(
             spacers,
             left_enzyme_inverted,
             right_enzyme_inverted,
+            tm_func=lambda x: primer3_calc_tm(x, settings),
         )
     except ValueError as e:
         raise HTTPException(400, *e.args)
@@ -198,11 +221,17 @@ async def primer_design_ebic(
     template: PrimerDesignQuery,
     max_inside: int = Query(..., description='The maximum length of the inside edge of the EBIC primer.'),
     max_outside: int = Query(..., description='The maximum length of the outside edge of the EBIC primer.'),
+    target_tm: float = Query(
+        ..., description='The desired melting temperature for the hybridization part of the primer.'
+    ),
+    target_tm_tolerance: float = Query(
+        3, description='The tolerance for the desired melting temperature for the hybridization part of the primer.'
+    ),
 ):
     """Design primers for EBIC"""
     dseqr = read_dsrecord_from_json(template.sequence)
     location = template.location.to_biopython_location()
-    return {'primers': ebic_primers(dseqr, location, max_inside, max_outside)}
+    return {'primers': ebic_primers(dseqr, location, max_inside, max_outside, target_tm, target_tm_tolerance)}
 
 
 # @router.post('/primer_design/gateway_attB', response_model=PrimerDesignResponse)
@@ -240,32 +269,6 @@ async def primer_design_ebic(
 #     return {'primers': primers}
 
 
-class ThermodynamicResult(BaseModel):
-    melting_temperature: float
-    deltaG: float
-    figure: str | None
-
-    @classmethod
-    def from_binding(cls, result):
-        return cls(
-            melting_temperature=result.tm,
-            deltaG=result.dg,
-            figure='\n'.join(result.ascii_structure_lines),
-        )
-
-
-def get_sequence_thermodynamic_result(sequences: list[str], method: callable) -> ThermodynamicResult | None:
-    """Get the thermodynamic result for a sequence, if the sequence is longer than primer3 60bp limit, it will be split into two
-    and the result with the lowest deltaG will be returned."""
-    results = [method(seq, output_structure=True) for seq in sequences]
-    results = [r for r in results if r.structure_found]
-    if len(results) == 0:
-        return None
-
-    result = min(results, key=lambda r: r.dg)
-    return ThermodynamicResult.from_binding(result)
-
-
 class PrimerDetailsResponse(BaseModel):
     melting_temperature: float
     gc_content: float
@@ -273,20 +276,20 @@ class PrimerDetailsResponse(BaseModel):
     hairpin: ThermodynamicResult | None
 
 
-@router.get('/primer_details', response_model=PrimerDetailsResponse)
+@router.post('/primer_details', response_model=PrimerDetailsResponse)
 async def primer_details(
-    sequence: str = Query(..., description='Primer sequence', pattern=r'^[ACGTacgt]+$'),
+    sequence: str = Body(..., description='Primer sequence', pattern=r'^[ACGTacgt]+$'),
+    settings: PrimerDesignSettings = Body(
+        ..., description='Primer design settings.', default_factory=PrimerDesignSettings
+    ),
 ):
     """Get information about a primer"""
     sequence = sequence.upper()
-    tm = bindings.calc_tm(sequence)
+    tm = primer3_calc_tm(sequence, settings)
     gc_content = gc_fraction(sequence)
 
-    thermodynamic_sequences = [sequence]
-    if len(sequence) > 60:
-        thermodynamic_sequences = [sequence[:60], sequence[-60:]]
-    homodimer = get_sequence_thermodynamic_result(thermodynamic_sequences, bindings.calc_homodimer)
-    hairpin = get_sequence_thermodynamic_result(thermodynamic_sequences, bindings.calc_hairpin)
+    homodimer = primer3_calc_homodimer(sequence, settings)
+    hairpin = primer3_calc_hairpin(sequence, settings)
 
     return {
         'melting_temperature': tm,
@@ -296,22 +299,14 @@ async def primer_details(
     }
 
 
-@router.get('/primer_heterodimer', response_model=ThermodynamicResult | None)
+@router.post('/primer_heterodimer', response_model=ThermodynamicResult | None)
 async def primer_heterodimer(
-    sequence1: str = Query(..., description='First primer sequence', pattern=r'^[ACGTacgt]+$'),
-    sequence2: str = Query(..., description='Second primer sequence', pattern=r'^[ACGTacgt]+$'),
+    sequence1: str = Body(..., description='First primer sequence', pattern=r'^[ACGTacgt]+$'),
+    sequence2: str = Body(..., description='Second primer sequence', pattern=r'^[ACGTacgt]+$'),
+    settings: PrimerDesignSettings = Body(
+        ..., description='Primer design settings.', default_factory=PrimerDesignSettings
+    ),
 ):
     """Get information about a primer pair"""
 
-    if len(sequence1) <= 60 or len(sequence2) <= 60:
-        sequence_pairs = [(sequence1, sequence2)]
-    else:
-        sequence_pairs = list(product((sequence1[:60], sequence1[-60:]), (sequence2[:60], sequence2[-60:])))
-
-    results = [bindings.calc_heterodimer(s1, s2, output_structure=True) for s1, s2 in sequence_pairs]
-    results = [r for r in results if r.structure_found]
-    if len(results) == 0:
-        return None
-
-    result = min(results, key=lambda r: r.dg)
-    return ThermodynamicResult.from_binding(result)
+    return primer3_calc_heterodimer(sequence1, sequence2, settings)

{opencloning-0.4.6 → opencloning-0.4.7}/src/opencloning/ncbi_requests.py

@@ -110,7 +110,13 @@ async def get_genbank_sequence(sequence_accession, start=None, end=None, strand=
 
     resp = await async_get(url, headers=headers, params=params)
     if resp.status_code == 200:
-        return pydna_parse(resp.text)[0]
+        try:
+            return pydna_parse(resp.text)[0]
+        except Exception:
+            # Now the ncbi returns something like this:
+            # Example: https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=nuccore&id=blah&rettype=gbwithparts&retmode=text
+            # 'Error: F a i l e d  t o  u n d e r s t a n d  i d :  b l a h '
+            raise HTTPException(404, 'wrong sequence accession')
     elif resp.status_code == 400:
         raise HTTPException(404, 'wrong sequence accession')
     elif resp.status_code == 503:

opencloning-0.4.7/src/opencloning/primer3_functions.py

@@ -0,0 +1,109 @@
+"""
+Functions to calculate primer melting temperature using primer3.
+primer3 should not be imported anywhere else.
+"""
+
+from pydantic import BaseModel, Field
+from itertools import product
+from primer3.bindings import (
+    calc_tm as _calc_tm,
+    design_primers as _design_primers,
+    calc_homodimer as _calc_homodimer,
+    calc_hairpin as _calc_hairpin,
+    calc_heterodimer as _calc_heterodimer,
+)
+
+
+class PrimerDesignSettings(BaseModel):
+    primer_dna_conc: float = Field(50, description='The DNA concentration in the primer solution (nM).')
+    primer_salt_monovalent: float = Field(
+        50, description='The monovalent salt concentration in the primer solution (mM).'
+    )
+    primer_salt_divalent: float = Field(
+        1.5, description='The divalent salt concentration in the primer solution (mM).'
+    )
+
+    def to_primer3_args(self) -> dict:
+        """Convert the settings to primer3 arguments."""
+        return {
+            'dna_conc': self.primer_dna_conc,
+            'mv_conc': self.primer_salt_monovalent,
+            'dv_conc': self.primer_salt_divalent,
+        }
+
+
+def primer3_calc_tm(seq: str, settings: PrimerDesignSettings) -> float:
+    return _calc_tm(seq.upper(), **settings.to_primer3_args())
+
+
+def primer3_design_primers(seq: str, seq_args: dict, global_args: dict):
+    report = _design_primers(
+        seq_args={
+            'SEQUENCE_ID': 'MH1000',
+            'SEQUENCE_TEMPLATE': seq,
+            **seq_args,
+        },
+        global_args=global_args,
+    )
+    return report
+
+
+class ThermodynamicResult(BaseModel):
+    melting_temperature: float
+    deltaG: float
+    figure: str | None
+
+    @classmethod
+    def from_binding(cls, result):
+        return cls(
+            melting_temperature=result.tm,
+            deltaG=result.dg,
+            figure='\n'.join(result.ascii_structure_lines),
+        )
+
+
+def get_sequence_thermodynamic_result(sequence: str, method: callable) -> ThermodynamicResult | None:
+    """Get the thermodynamic result for a sequence, if the sequence is longer than primer3 60bp limit, it will be split into two
+    and the result with the lowest deltaG will be returned."""
+    sequence = sequence.upper()
+    if len(sequence) <= 60:
+        sequences = [sequence]
+    else:
+        sequences = [sequence[:60], sequence[-60:]]
+
+    results = [method(seq) for seq in sequences]
+    results = [r for r in results if r.structure_found]
+    if len(results) == 0:
+        return None
+
+    result = min(results, key=lambda r: r.dg)
+    return ThermodynamicResult.from_binding(result)
+
+
+def primer3_calc_homodimer(seq: str, settings: PrimerDesignSettings) -> ThermodynamicResult:
+    return get_sequence_thermodynamic_result(
+        seq, lambda x: _calc_homodimer(x, output_structure=True, **settings.to_primer3_args())
+    )
+
+
+def primer3_calc_hairpin(seq: str, settings: PrimerDesignSettings) -> ThermodynamicResult:
+    return get_sequence_thermodynamic_result(
+        seq, lambda x: _calc_hairpin(x, output_structure=True, **settings.to_primer3_args())
+    )
+
+
+def primer3_calc_heterodimer(seq1: str, seq2: str, settings: PrimerDesignSettings) -> ThermodynamicResult:
+    if len(seq1) <= 60 or len(seq2) <= 60:
+        sequence_pairs = [(seq1, seq2)]
+    else:
+        sequence_pairs = list(product((seq1[:60], seq1[-60:]), (seq2[:60], seq2[-60:])))
+
+    results = [
+        _calc_heterodimer(s1, s2, output_structure=True, **settings.to_primer3_args()) for s1, s2 in sequence_pairs
+    ]
+    results = [r for r in results if r.structure_found]
+    if len(results) == 0:
+        return None
+
+    result = min(results, key=lambda r: r.dg)
+    return ThermodynamicResult.from_binding(result)

{opencloning-0.4.6 → opencloning-0.4.7}/src/opencloning/primer_design.py

@@ -7,13 +7,8 @@ from .pydantic_models import PrimerModel
 from Bio.Seq import reverse_complement
 from Bio.Restriction.Restriction import RestrictionType
 from Bio.Data.IUPACData import ambiguous_dna_values as _ambiguous_dna_values
-from primer3.bindings import calc_tm as _calc_tm
 from typing import Callable
-
-
-def primer3_calc_tm(seq: str) -> float:
-    return _calc_tm(seq.upper())
-
+from .primer3_functions import primer3_calc_tm, PrimerDesignSettings
 
 ambiguous_dna_values = _ambiguous_dna_values.copy()
 # Remove acgt
@@ -21,6 +16,10 @@ for base in 'ACGT':
     del ambiguous_dna_values[base]
 
 
+def default_tm_func(sequence: str) -> float:
+    return primer3_calc_tm(sequence, PrimerDesignSettings())
+
+
 def homologous_recombination_primers(
     pcr_seq: Dseqrecord,
     pcr_loc: SimpleLocation,
@@ -31,7 +30,7 @@ def homologous_recombination_primers(
     insert_forward: bool,
     target_tm: float,
     spacers: list[str] | None = None,
-    tm_func: Callable[[str], float] = primer3_calc_tm,
+    tm_func: Callable[[str], float] = default_tm_func,
     estimate_function: Callable[[str], float] | None = None,
 ) -> tuple[str, str]:
 
@@ -91,7 +90,7 @@ def gibson_assembly_primers(
     target_tm: float,
     circular: bool,
     spacers: list[str] | None = None,
-    tm_func: Callable[[str], float] = primer3_calc_tm,
+    tm_func: Callable[[str], float] = default_tm_func,
     estimate_function: Callable[[str], float] | None = None,
 ) -> list[PrimerModel]:
 
@@ -157,7 +156,7 @@ def simple_pair_primers(
     spacers: list[str] | None = None,
     left_enzyme_inverted: bool = False,
     right_enzyme_inverted: bool = False,
-    tm_func: Callable[[str], float] = primer3_calc_tm,
+    tm_func: Callable[[str], float] = default_tm_func,
     estimate_function: Callable[[str], float] | None = None,
 ) -> tuple[PrimerModel, PrimerModel]:
     """

opencloning-0.4.6/src/opencloning/batch_cloning/EBIC/barcode.gb

@@ -1,23 +0,0 @@
-LOCUS       barcode         151 bp DNA     linear   UNA 15-SEP-2024
-DEFINITION  natural linear DNA
-ACCESSION   .
-VERSION     .
-KEYWORDS    .
-SOURCE      natural DNA sequence
-  ORGANISM  unspecified
-REFERENCE   1  (bases 1 to 151)
-  AUTHORS   SnapGene License
-  TITLE     Direct Submission
-  JOURNAL   Exported Dec 20, 2024 from SnapGene 7.2.1
-            https://www.snapgene.com
-FEATURES             Location/Qualifiers
-     source          1..151
-                     /mol_type="genomic DNA"
-                     /organism="unspecified"
-     misc_feature    15..137
-                     /label=CG_Barcode1
-ORIGIN
-        1 ataggtctct aatggctctt attcgggaca tttacaatgc cacccactgg gagtgattat
-       61 gagtctcatt gagactgctc accacagcat gaattctctg gttcggtggc gcatagctat
-      121 cgctgttcct gccactggct tagagaccta t
-//

opencloning-0.4.6/src/opencloning/batch_cloning/EBIC/common_plasmid.gb

@@ -1,218 +0,0 @@
-LOCUS       KanSacB+Scarlett        5747 bp DNA     circular SYN 08-AUG-2024
-DEFINITION  synthetic circular DNA
-ACCESSION   .
-VERSION     .
-KEYWORDS    .
-SOURCE      synthetic DNA construct
-  ORGANISM  recombinant plasmid
-REFERENCE   1  (bases 1 to 5747)
-  AUTHORS   SnapGene License
-  TITLE     Direct Submission
-  JOURNAL   Exported Dec 20, 2024 from SnapGene 7.2.1
-            https://www.snapgene.com
-FEATURES             Location/Qualifiers
-     source          1..5747
-                     /mol_type="other DNA"
-                     /organism="recombinant plasmid"
-     misc_feature    1..4
-                     /label=AATG
-                     /note="/color=#f7e8f7"
-     misc_feature    5..2586
-                     /label=From pC0.026
-                     /note="/color=white"
-                     /note="/indicates_part=True"
-                     /note="/source=pC0.026"
-     promoter        5..450
-                     /gene="sacR"
-                     /label=sacB promoter
-                     /note="sacB promoter and control region"
-     CDS             451..1872
-                     /codon_start=1
-                     /gene="Bacillus subtilis sacB"
-                     /product="secreted levansucrase that renders
-                     bacterialgrowth sensitive to sucrose"
-                     /label=SacB
-                     /note="negative selection marker"
-                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
-                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
-                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
-                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
-                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
-                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
-                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
-                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
-                     VKDSILEQGQLTVNK"
-     misc_feature    2587..2590
-                     /label=AGGT
-                     /note="/color=#f7e8f7"
-     misc_feature    2591..3800
-                     /label=From pC0.027
-                     /note="/color=white"
-                     /note="/indicates_part=True"
-                     /note="/source=pC0.027"
-     CDS             complement(2875..3690)
-                     /codon_start=1
-                     /gene="aph(3')-Ia"
-                     /product="aminoglycoside phosphotransferase"
-                     /label=KanR
-                     /note="confers resistance to kanamycin in bacteria or
-                     G418(Geneticin(R)) in eukaryotes"
-                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
-                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
-                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
-                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
-                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
-     misc_feature    3691..3800
-                     /label=promoter
-     misc_feature    3801..3804
-                     /label=GCTT
-                     /note="/color=#f7e8f7"
-     misc_feature    3805..4682
-                     /label=From gblock_OriTOriR
-                     /note="/color=white"
-                     /note="/indicates_part=True"
-                     /note="/source=gblock_OriTOriR"
-     misc_feature    4675..4681
-                     /label=SapI site
-     promoter        4713..4842
-                     /label=BBa_J72163 GlpT Promoter
-                     /note="label: BBa_J72163 GlpT Promoter"
-     primer_bind     4713..4732
-                     /label=CG17
-     RBS             4843..4878
-                     /label=RBS
-                     /note="label: RBS"
-     RBS             4861..4872
-                     /label=RBS
-                     /note="note: strong bacterial ribosome binding site
-                     (Elowitz and Leibler, 2000)"
-     CDS             4879..5577
-                     /label=mScarlet
-                     /note="codon_start: 1 label: mScarlet transl_table: 1
-                     translation:
-                     MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSW
-                     DILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGT
-                     LIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLAD
-                     FKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTGGMDELYK*"
-     terminator      5578..5706
-                     /label=BBa_B0015 Terminator
-                     /note="label: BBa_B0015 Terminator"
-     terminator      5586..5657
-                     /label=rrnB T1 terminator
-                     /note="label: rrnB T1 terminator note: pLannotate note:
-                     /database=snapgene note: /fragment=False note:
-                     /identity=100.0 note: /match_length=100.0 note:
-                     /other=terminator"
-     terminator      5673..5700
-                     /label=T7Te terminator
-                     /note="label: T7Te terminator note: pLannotate note:
-                     /database=snapgene note: /fragment=False note:
-                     /identity=100.0 note: /match_length=100.0 note:
-                     /other=terminator"
-     primer_bind     complement(5685..5706)
-                     /label=CG18
-     misc_feature    5734..5747
-                     /label=From gblock_OriTOriR
-                     /note="/color=white"
-                     /note="/indicates_part=True"
-                     /note="/source=gblock_OriTOriR"
-     misc_feature    5738..5744
-                     /label=SapI site
-ORIGIN
-        1 aatgcacata tacctgccgt tcactattat ttagtgaaat gagatattat gatattttct
-       61 gaattgtgat taaaaaggca actttatgcc catgcaacag aaactataaa aaatacagag
-      121 aatgaaaaga aacagataga ttttttagtt ctttaggccc gtagtctgca aatcctttta
-      181 tgattttcta tcaaacaaaa gaggaaaata gaccagttgc aatccaaacg agagtctaat
-      241 agaatgaggt cgaaaagtaa atcgcgcggg tttgttactg ataaagcagg caagacctaa
-      301 aatgtgtaaa gggcaaagtg tatactttgg cgtcacccct tacatatttt aggtcttttt
-      361 ttattgtgcg taactaactt gccatcttca aacaggaggg ctggaagaag cagaccgcta
-      421 acacagtaca taaaaaagga gacatgaacg atgaacatca aaaagtttgc aaaacaagca
-      481 acagtattaa cctttactac cgcactgctg gcaggaggcg caactcaagc gtttgcgaaa
-      541 gaaacgaacc aaaagccata taaggaaaca tacggcattt cccatattac acgccatgat
-      601 atgctgcaaa tccctgaaca gcaaaaaaat gaaaaatatc aagttcctga attcgattcg
-      661 tccacaatta aaaatatctc ttctgcaaaa ggcctggacg tttgggacag ctggccatta
-      721 caaaacgctg acggcactgt cgcaaactat cacggctacc acatcgtctt tgcattagcc
-      781 ggagatccta aaaatgcgga tgacacatcg atttacatgt tctatcaaaa agtcggcgaa
-      841 acttctattg acagctggaa aaacgctggc cgcgtcttta aagacagcga caaattcgat
-      901 gcaaatgatt ctatcctaaa agaccaaaca caagaatggt caggttcagc cacatttaca
-      961 tctgacggaa aaatccgttt attctacact gatttctccg gtaaacatta cggcaaacaa
-     1021 acactgacaa ctgcacaagt taacgtatca gcatcagaca gctctttgaa catcaacggt
-     1081 gtagaggatt ataaatcaat ctttgacggt gacggaaaaa cgtatcaaaa tgtacagcag
-     1141 ttcatcgatg aaggcaacta cagctcaggc gacaaccata cgctgagaga tcctcactac
-     1201 gtagaagata aaggccacaa atacttagta tttgaagcaa acactggaac tgaagatggc
-     1261 taccaaggcg aagaatcttt atttaacaaa gcatactatg gcaaaagcac atcattcttc
-     1321 cgtcaagaaa gtcaaaaact tctgcaaagc gataaaaaac gcacggctga gttagcaaac
-     1381 ggcgctctcg gtatgattga gctaaacgat gattacacac tgaaaaaagt gatgaaaccg
-     1441 ctgattgcat ctaacacagt aacagatgaa attgaacgcg cgaacgtctt taaaatgaac
-     1501 ggcaaatggt acctgttcac tgactcccgc ggatcaaaaa tgacgattga cggcattacg
-     1561 tctaacgata tttacatgct tggttatgtt tctaattctt taactggccc atacaagccg
-     1621 ctgaacaaaa ctggccttgt gttaaaaatg gatcttgatc ctaacgatgt aacctttact
-     1681 tactcacact tcgctgtacc tcaagcgaaa ggaaacaatg tcgtgattac aagctatatg
-     1741 acaaacagag gattctacgc agacaaacaa tcaacgtttg cgccaagctt cctgctgaac
-     1801 atcaaaggca agaaaacatc tgttgtcaaa gacagcatcc ttgaacaagg acaattaaca
-     1861 gttaacaaat aaaaacgcaa aagaaaatgc cgatatccta ttggcatttt cttttatttc
-     1921 ttatcaacat aaaggtgaat cccatatgaa ctatataaaa gcaggcaaat ggctaaccgt
-     1981 attcctaacc ttttggtaat gactccaact tattgatagt gttttatgtt cagataatgc
-     2041 ccgatgactt tgtcatgcag ctccaccgat tttgagaacg acagcgactt ccgtcccagc
-     2101 cgtgccaggt gctgcctcag attcaggtta tgccgctcaa ttcgctgcgt atatcgcttg
-     2161 ctgattacgt gcagctttcc cttcaggcgg gattcataca gcggccagcc atccgtcatc
-     2221 catatcacca cgtcaaaggg tgacagcagg ctcataagac gccccagcgt cgccatagtg
-     2281 cgttcaccga atacgtgcgc aacaaccgtc taccggagac tgtcatacgc gtaaaacagc
-     2341 cagcgctggc gcgatttagc cccgacatag ccccactgtt cgtccatttc cgcgcagacg
-     2401 atgacgtcac tgcccggctg tatgcgcgag gttaccgact gcggcctgag ttttttaagt
-     2461 gacgtaaaat cgtgttgagg ccaacgccca taatgcgggc tgttgcccgg catccaacgc
-     2521 cattcatggc catatcaatg attttctggt gcgtaccggg ttgagaagcg gtgtaagtga
-     2581 actgcaaggt ctgaggtctg cctcgtgaag aaggtgttgc tgactcatac caggcctgaa
-     2641 tcgccccatc atccagccag aaagtgaggg agccacggtt gatgagagct ttgttgtagg
-     2701 tggaccagtt ggtgattttg aacttttgct ttgccacgga acggtctgcg ttgtcgggaa
-     2761 gatgcgtgat ctgatccttc aactcagcaa aagttcgatt tattcaacaa agccgccgtc
-     2821 ccgtcaagtc agcgtaatgc tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa
-     2881 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
-     2941 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
-     3001 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
-     3061 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
-     3121 cggtgagaat ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt
-     3181 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
-     3241 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
-     3301 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
-     3361 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
-     3421 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
-     3481 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
-     3541 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
-     3601 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga
-     3661 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
-     3721 agacagtttt attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt
-     3781 ttgagacaca acgtggcttt gcttgtacac cttgataggt gggctgccct tcctggttgg
-     3841 cttggtttca tcagccatcc gcttgccctc atctgttacg ccggcggtag ccggccagcc
-     3901 tcgcagagca ggattcccgt tgagcaccgc caggtgcgaa taagggacag tgaagaagga
-     3961 acacccgctc gcgggtgggc ctacttcacc tatcctgccc ggctgacgcc gttggataca
-     4021 ccaaggaaag tctacacgaa ccctttggca aaatatcaaa ggatcttctt gagatccttt
-     4081 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
-     4141 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
-     4201 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
-     4261 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
-     4321 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
-     4381 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
-     4441 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
-     4501 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
-     4561 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
-     4621 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggcgctctt
-     4681 catgctcgga gagagacctt tgccttgtct tcgaaagtga aacgtgattt catgcgtcat
-     4741 tttgaacatt ttgtaaatct tatttaataa tgtgtgcggc aattcacatt taatttatga
-     4801 atgttttctt aacatcgcgg caactcaaga aacggcaggt tcggatctta gctactagag
-     4861 aaagaggaga aatactagat ggttagcaaa ggcgaggcgg ttatcaagga gtttatgcgt
-     4921 tttaaggttc acatggaggg tagcatgaac ggtcacgagt tcgagatcga gggtgaaggc
-     4981 gagggtcgtc cgtacgaagg cacccagacc gcgaagctga aagtgaccaa gggtggcccg
-     5041 ctgccgttca gctgggacat cctgagcccg cagttcatgt atggcagccg tgcgtttacc
-     5101 aaacacccgg cggacattcc ggattactat aagcaaagct tcccggaagg ttttaaatgg
-     5161 gagcgtgtta tgaacttcga agatggtggc gcggtgaccg ttacccagga caccagcctg
-     5221 gaggatggca ccctgattta caaggtgaaa ctgcgtggca ccaactttcc gccggatggt
-     5281 ccggttatgc agaagaaaac gatgggttgg gaagcgagca ccgagcgtct gtatccggaa
-     5341 gatggcgtgc tgaagggtga tatcaaaatg gcgctgcgtc tgaaggacgg tggccgttac
-     5401 ctggcggatt ttaagaccac ctataaagcg aagaaaccgg tgcaaatgcc gggtgcgtac
-     5461 aacgttgacc gtaaactgga tattaccagc cacaacgagg attataccgt ggttgagcaa
-     5521 tatgagcgta gcgagggtcg ccacagcacc ggcggcatgg acgaactgta taagtgacca
-     5581 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
-     5641 tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg
-     5701 tttatagaag acaagggaaa ggtctctcgc tgagatagaa gagcacg
-//

opencloning-0.4.6/src/opencloning/batch_cloning/EBIC/example.py

@@ -1,180 +0,0 @@
-import asyncio
-import os
-from primer3 import bindings
-import json
-from fastapi import UploadFile, Response
-
-from opencloning.pydantic_models import (
-    GenomeCoordinatesSource,
-    TextFileSequence,
-    PrimerModel,
-    PCRSource,
-    RestrictionAndLigationSource,
-    BaseCloningStrategy,
-    HomologousRecombinationSource,
-)
-from opencloning.batch_cloning.EBIC.primer_design_settings import amanda_settings
-from opencloning.endpoints.external_import import genome_coordinates, read_from_file
-from opencloning.endpoints.assembly import pcr, restriction_and_ligation, homologous_recombination
-from opencloning.dna_functions import read_dsrecord_from_json
-
-# Settings for design
-padding = 1000
-max_inside = 20
-max_outside = 70
-outside_edge = padding - max_outside
-inside_edge = padding + max_inside
-
-adapter_left_fwd = 'ataGGTCTCtGGAG'
-adapter_left_rvs = 'ataGGTCTCtCATT'
-adapter_right_fwd = 'ataGGTCTCtGCTT'
-adapter_right_rvs = 'ataGGTCTCtAGCG'
-
-
-def design_primers_ebic(seq: str, seq_args: dict):
-
-    report = bindings.design_primers(
-        seq_args={
-            'SEQUENCE_ID': 'MH1000',
-            'SEQUENCE_TEMPLATE': seq,
-            **seq_args,
-        },
-        global_args=amanda_settings,
-    )
-
-    return report
-
-
-async def main():
-    cloning_strategy = BaseCloningStrategy(
-        sequences=[],
-        sources=[],
-        primers=[],
-        description='EBIC cloning strategy',
-    )
-
-    initial_source = GenomeCoordinatesSource(
-        id=0,
-        assembly_accession='GCF_002847425.1',
-        sequence_accession='NZ_CP025534.1',
-        start=2700777 - padding,
-        end=2701295 + padding,
-        strand=1,
-    )
-
-    if os.path.exists('cached_response.json'):
-        with open('cached_response.json', 'r') as f:
-            locus = json.load(f)
-            locus['sequences'][0] = TextFileSequence.model_validate(locus['sequences'][0])
-            locus['sources'][0] = GenomeCoordinatesSource.model_validate(locus['sources'][0])
-    else:
-        print('No cached response found, fetching from server')
-        locus = await genome_coordinates(initial_source)
-        with open('cached_response.json', 'w') as f:
-            json.dump(
-                {
-                    'sequences': [locus['sequences'][0].model_dump()],
-                    'sources': [locus['sources'][0].model_dump()],
-                },
-                f,
-            )
-
-    locus_seq: TextFileSequence = locus['sequences'][0]
-    locus_source: GenomeCoordinatesSource = locus['sources'][0]
-    locus_dseqr = read_dsrecord_from_json(locus_seq)
-
-    cloning_strategy.add_source_and_sequence(locus_source, locus_seq)
-
-    left_template = locus_dseqr[:inside_edge]
-    right_template = locus_dseqr[-inside_edge:]
-
-    seq_args_left = {
-        'SEQUENCE_PRIMER_PAIR_OK_REGION_LIST': f'0,{int(padding/2)},{outside_edge},{max_outside + max_inside}',
-    }
-    seq_args_right = {
-        'SEQUENCE_PRIMER_PAIR_OK_REGION_LIST': f'0,{max_outside + max_inside},{len(right_template) - int(padding/2)},{int(padding/2)}',
-    }
-
-    report_left = design_primers_ebic(str(left_template.seq), seq_args_left)
-    report_right = design_primers_ebic(str(right_template.seq), seq_args_right)
-
-    primer_names = ['left_fwd', 'left_rvs', 'right_fwd', 'right_rvs']
-    primer_seqs = [
-        adapter_left_fwd + report_left['PRIMER_LEFT'][0]['SEQUENCE'],
-        adapter_left_rvs + report_left['PRIMER_RIGHT'][0]['SEQUENCE'],
-        adapter_right_fwd + report_right['PRIMER_LEFT'][0]['SEQUENCE'],
-        adapter_right_rvs + report_right['PRIMER_RIGHT'][0]['SEQUENCE'],
-    ]
-    primers = [PrimerModel(id=0, name=primer_names[i], sequence=primer_seqs[i]) for i in range(4)]
-    for primer in primers:
-        cloning_strategy.add_primer(primer)
-
-    pcr_left_source = PCRSource(id=0, output_name='left_homology_arm')
-    resp = await pcr(pcr_left_source, [locus_seq], [primers[0], primers[1]], 17, 0)
-    pcr_product_left: TextFileSequence = resp['sequences'][0]
-    pcr_left_source: PCRSource = resp['sources'][0]
-    cloning_strategy.add_source_and_sequence(pcr_left_source, pcr_product_left)
-
-    pcr_right_source = PCRSource(id=0, output_name='right_homology_arm')
-    resp = await pcr(pcr_right_source, [locus_seq], [primers[2], primers[3]], 17, 0)
-    pcr_product_right: TextFileSequence = resp['sequences'][0]
-    pcr_right_source: PCRSource = resp['sources'][0]
-    cloning_strategy.add_source_and_sequence(pcr_right_source, pcr_product_right)
-
-    with open(os.path.join(os.path.dirname(__file__), 'barcode.gb'), 'rb') as f:
-        dummy_resp = Response()
-        resp = await read_from_file(
-            dummy_resp, UploadFile(file=f, filename='barcode.gb'), None, None, True, 'barcode', None, None
-        )
-
-    barcode_source = resp['sources'][0]
-    barcode_seq: TextFileSequence = resp['sequences'][0]
-    cloning_strategy.add_source_and_sequence(barcode_source, barcode_seq)
-
-    with open(os.path.join(os.path.dirname(__file__), 'common_plasmid.gb'), 'rb') as f:
-        dummy_resp = Response()
-        resp = await read_from_file(
-            dummy_resp,
-            UploadFile(file=f, filename='common_plasmid.gb'),
-            None,
-            None,
-            True,
-            'common_plasmid',
-            None,
-            None,
-        )
-
-    common_plasmid_source = resp['sources'][0]
-    common_plasmid_seq: TextFileSequence = resp['sequences'][0]
-    cloning_strategy.add_source_and_sequence(common_plasmid_source, common_plasmid_seq)
-
-    golgen_gate = RestrictionAndLigationSource(id=0, output_name='golgen_gate', restriction_enzymes=['BsaI'])
-    resp = await restriction_and_ligation(
-        golgen_gate, [pcr_product_left, pcr_product_right, barcode_seq, common_plasmid_seq], False, True
-    )
-    golgen_gate_product: TextFileSequence = resp['sequences'][0]
-    golgen_gate_source: RestrictionAndLigationSource = resp['sources'][0]
-    cloning_strategy.add_source_and_sequence(golgen_gate_source, golgen_gate_product)
-
-    homologous_recombination_source = HomologousRecombinationSource(id=0, output_name='engineered_allele')
-    resp = await homologous_recombination(homologous_recombination_source, [locus_seq, golgen_gate_product], 17)
-
-    multi_site_sources = [
-        i for i, s in enumerate(resp['sources']) if all(join.left_location != join.right_location for join in s.input)
-    ]
-    if len(multi_site_sources) > 1:
-        raise ValueError('Multiple insertions possible')
-
-    possible_sources = [resp['sources'][i] for i in multi_site_sources]
-    possible_sequences = [resp['sequences'][i] for i in multi_site_sources]
-    homologous_recombination_product: TextFileSequence = possible_sequences[0]
-    homologous_recombination_source: HomologousRecombinationSource = possible_sources[0]
-    cloning_strategy.add_source_and_sequence(homologous_recombination_source, homologous_recombination_product)
-
-    # Save cloning strategy to JSON file
-    with open('cloning_strategy.json', 'w') as f:
-        json.dump(cloning_strategy.model_dump(), f, indent=2)
-    return cloning_strategy
-
-
-asyncio.run(main())

opencloning-0.4.6/src/opencloning/batch_cloning/EBIC/primer_design_settings.py

@@ -1,38 +0,0 @@
-amanda_settings = {
-    # Tm: Optimal 60-62°C, but this can be extended to 58-64°C if necessary.
-    # All primers need to ideally be within 5°C to ensure they can be run on the same plate for high-throughput.
-    'PRIMER_OPT_TM': 61,  # Optimal Tm
-    'PRIMER_MIN_TM': 58,  # Minimum Tm
-    'PRIMER_MAX_TM': 64,  # Maximum Tm
-    'PRIMER_PAIR_MAX_DIFF_TM': 5,  # Maximum Tm difference within a pair
-    # Salt concentration: 50mM NaCl, 1.5mM MgCl2
-    'PRIMER_SALT_MONOVALENT': 50.0,
-    'PRIMER_SALT_DIVALENT': 1.5,
-    # 500 nM primer concentration
-    'PRIMER_DNA_CONC': 500.0,
-    # Primer size: 17-30nt.
-    'PRIMER_MIN_SIZE': 17,  # Minimum primer length
-    'PRIMER_MAX_SIZE': 30,  # Maximum primer length
-    'PRIMER_OPT_SIZE': 22,  # Typical optimal size (can be adjusted)
-    # GC content: between 35 and 65%.
-    'PRIMER_MIN_GC': 35,  # Minimum GC content percentage
-    'PRIMER_MAX_GC': 65,  # Maximum GC content percentage
-    # A 1 or 2nt GC clamp at the 3’ end of the primer helps with PCR efficiency.
-    'PRIMER_GC_CLAMP': 1,  # Number of GC bases required at the 3’ end
-    # Product size: ~950bp homologous arms, though this can be shortened if necessary.
-    'PRIMER_PRODUCT_SIZE_RANGE': [[800, 1000]],  # Product size range in base pairs
-    # Avoid primers with long repetitions of nucleotides in a row (e.g., TTTTT).
-    'PRIMER_MAX_POLY_X': 4,  # Maximum allowable repetitions of a single nucleotide
-    # Dimer and hairpin structure considerations:
-    # Self-dimers – avoid ΔG below -10
-    # Heterodimers – avoid ΔG below -10
-    # Hairpin loops - avoid ΔG below -5
-    'PRIMER_MAX_SELF_ANY': 47,
-    'PRIMER_MAX_SELF_END': 47,
-    'PRIMER_MAX_HAIRPIN': 47,
-    # Return only one primer pair
-    'PRIMER_NUM_RETURN': 1,
-}
-
-# Avoid primers with homology to other sites in the genome.
-# This can be checked using external tools like BLAST for specificity.