给 Agent 出一道高价值分子克隆题,
并审判最强 Agent 的产出

cd /Users/maxue/Documents/简历/kimi/question/inputs/ && ls -la && echo "---KRAS---" && head -5 kras_ENST00000256078.fa && echo "---GB head---" && head -40 plvx-egfp-ires-puro.gb

total 72
drwxr-xr-x@ 4 maxue  staff    128 Jun 23 17:12 .
drwxr-xr-x@ 4 maxue  staff    128 Jun 23 17:17 ..
-rw-r--r--@ 1 maxue  staff   5609 Jun 23 17:12 kras_ENST00000256078.fa
-rw-------@ 1 maxue  staff  26489 Jun 23 17:10 plvx-egfp-ires-puro.gb
---KRAS---
>ENST00000256078.10 KRAS-201 cdna:protein_coding (full transcript: 5'UTR + CDS + 3'UTR)
CTAGGCGGCGGCCGCGGCGGCGGAGGCAGCAGCGGCGGCGGCAGTGGCGGCGGCGAAGGT
GGCGGCGGCTCGGCCAGTACTCCCGGCCCCCGCCATTTCGGACTGGGAGCGAGCGCGGCG
CAGGCACTGAAGGCGGCGGCGGGGCCAGAGGCTCAGCGGCTCCCAGGTGCGGGAGAGAGG
CCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAG
---GB head---
LOCUS       pLVX-EGFP-IRES-puro     8911 bp ds-DNA     circular     06-SEP-2025
DEFINITION  mammalian expression of EGFP with puromycin selection.
KEYWORDS    "accession:addgene_128652_320737"
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
COMMENT     Imported from database: AddGene Entry: pLVX-EGFP-IRES-puro URL:
            https://www.addgene.org/browse/sequence/320737/ GENERAL Depositing
            Lab: Robert Sobol Publication: Fang et al Nat Commun. 2014 Nov
            26;5:5513. doi: 10.1038/ncomms6513. Purpose: mammalian expression
            of EGFP with puromycin selection BACKBONE Backbone size w/o insert
            (bp): 8247 Selectable markers: Puromycin Total vector size (bp):
            8958 Vector backbone: pLVX-GWE-IRES-puro Vector type: Mammalian
            Expression, Lentiviral CLONING INFORMATION 3′ sequencing primer:
            M13 reverse for pENTR vector 5′ sequencing primer: M13 forward for
            pENTR vector Cloning method: Gateway Cloning GROWTH IN BACTERIA
            Bacterial Resistance(s): Ampicillin, 100 μg/mL Copy number: Unknown
            Growth Strain(s): NEB Stable Growth Temperature: 37°C GENE/INSERT
            Alt name: N/A GenBank ID:  Gene/Insert name: EGFP Insert Size (bp):
            720 Mutation: Wild type Promoter: CMV Tag / Fusion Protein:  TERMS
            AND LICENSES Academic/Nonprofit Terms:  Industry Terms:
FEATURES             Location/Qualifiers
     source          1..8911
                     /label="source"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..634
                     /label="3' LTR"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label="HIV-1 Psi"
                     /ApEinfo_revcolor="#75c6a9"
                     /ApEinfo_fwdcolor="#75c6a9"
                     /note="packaging signal of human immunodeficiency virus type 1"
     misc_feature    1303..1536
                     /label="RRE"
                     /ApEinfo_revcolor="#b7e6d7"

/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python -c "import labbench2.cloning as c; print([x for x in dir(c) if not x.startswith('_')])"

['CloningProtocol', 'accuracy_reward', 'cloning_digest_reward', 'cloning_execution_reward', 'cloning_format_reward', 'cloning_protocol', 'cloning_similarity_reward', 'compare_sequences', 'enzyme_cut', 'execution_reward', 'format_reward', 'gibson', 'goldengate', 'recursive_restriction_assemble', 'restriction_assemble', 'restriction_enzyme', 'rewards', 'sequence_alignment', 'sequence_models', 'similarity_reward', 'simulate_pcr', 'utils']

cd /Users/maxue/Documents/简历/kimi/question/inputs/ && grep -n -i "EGFP\|CDS\|IRES\|puro\|CMV" plvx-egfp-ires-puro.gb | head -40

1:LOCUS       pLVX-EGFP-IRES-puro     8911 bp ds-DNA     circular     06-SEP-2025
2:DEFINITION  mammalian expression of EGFP with puromycin selection.
6:COMMENT     Imported from database: AddGene Entry: pLVX-EGFP-IRES-puro URL:
10:            of EGFP with puromycin selection BACKBONE Backbone size w/o insert
11:            (bp): 8247 Selectable markers: Puromycin Total vector size (bp):
12:            8958 Vector backbone: pLVX-GWE-IRES-puro Vector type: Mammalian
18:            Alt name: N/A GenBank ID:  Gene/Insert name: EGFP Insert Size (bp):
19:            720 Mutation: Wild type Promoter: CMV Tag / Fusion Protein:  TERMS
43:     CDS             1721..1765
56:                     /label="CMV enhancer"
61:                     /label="CMV promoter"
64:                     /note="human cytomegalovirus (CMV) immediate early promoter"
66:                     /label="CMV-F"
69:                     /note="Human CMV immediate early promoter, forward primer"
74:                     /note="Human CMV promoter, forward primer"
82:     CDS             join(2869..2871,2872..2874,2875..3588)
83:                     /label="EGFP"
89:     CDS             join(2869..2871,2872..2874,2875..3585)
90:                     /label="EGFP"
96:                     /label="EGFP-N"
99:                     /note="EGFP, reverse primer"
104:                     /note="For distinguishing EGFP variants, reverse primer"
106:                     /label="EGFP-C"
109:                     /note="EGFP, forward primer"
118:                     /label="IRES2"
121:                     /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)"
123:                     /label="IRES reverse"
126:                     /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev"
128:                     /label="IRES-F"
131:                     /note="IRES internal ribosome entry site, forward primer"
133:                     /label="IRES2"
136:                     /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)"
138:                     /label="IRES2"
141:                     /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)"
143:                     /label="Puro-R"
146:                     /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R"
147:     CDS             4267..4866
148:                     /label="PuroR"
151:                     /note="confers resistance to puromycin"
153:                     /product="puromycin N-acetyltransferase"

cd /Users/maxue/Documents/简历/kimi/question/inputs/ && /Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python << 'EOF'
from Bio import SeqIO
gb = SeqIO.read("plvx-egfp-ires-puro.gb", "genbank")
seq = str(gb.seq)
print("len", len(seq))
# EGFP CDS 2869..3588 (1-based inclusive). Python idx: 2868..3588
egfp = seq[2868:3588]
print("EGFP start codon:", egfp[:3], "len", len(egfp), "div3", len(egfp)%3)
print("EGFP last 6:", egfp[-6:])
# Translate
from Bio.Seq import Seq
print("EGFP prot N:", str(Seq(egfp).translate())[:10], "... C:", str(Seq(egfp).translate())[-5:])
# context around EGFP start (upstream Kozak) and downstream
print("upstream of ATG (2820-2870):", seq[2820:2868])
print("ATG region:", seq[2865:2880])
print("downstream of stop (3588-3620):", seq[3588:3620])
EOF

len 8911
EGFP start codon: ATG len 720 div3 0
EGFP last 6: AAGTAA
EGFP prot N: MVSKGEELFT ... C: ELYK*
upstream of ATG (2820-2870): TCAACAAGTTTGTACAAAAAAGCAGGCTCCGCGGCCGCCCCCTTCACC
ATG region: ACCATGGTGAGCAAG
downstream of stop (3588-3620): AAGGGTGGGCGCGCCGACCCAGCTTTCTTGTA

cd /Users/maxue/Documents/简历/kimi/question/inputs/ && /Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python << 'EOF'
from Bio import SeqIO
from Bio.Seq import Seq
rec = SeqIO.read("kras_ENST00000256078.fa","fasta")
s = str(rec.seq)
print("transcript len", len(s))
# Find CDS: scan for longest ORF starting ATG. KRAS 4B protein is 188 aa.
# Find all ATG, look for the canonical start. KRAS CDS in this transcript.
import re
best=None
for m in re.finditer("ATG", s):
    i=m.start()
    sub=s[i:]
    sub=sub[:len(sub)//3*3]
    prot=str(Seq(sub).translate(to_stop=True))
    if best is None or len(prot)>best[2]:
        best=(i, prot, len(prot))
i,prot,L=best
print("longest ORF start idx",i,"prot len",L)
print("prot start:",prot[:15])
print("prot end:",prot[-15:])
cds = s[i:i+(L+1)*3]
print("CDS len", len(cds), "starts", cds[:9], "ends", cds[-6:])
print("translate ends w stop:", str(Seq(cds).translate())[-3:])
EOF

transcript len 5430
longest ORF start idx 190 prot len 189
prot start: MTEYKLVVVGAGGVG
prot end: EKTPGCVKIKKCIIM
CDS len 570 starts ATGACTGAA ends ATGTAA
translate ends w stop: IM*

cd /Users/maxue/Documents/简历/kimi/question/inputs/ && /Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python << 'EOF'
from Bio import SeqIO
from Bio.Seq import Seq
rec = SeqIO.read("kras_ENST00000256078.fa","fasta")
s=str(rec.seq)
cds=s[190:190+570]
prot=str(Seq(cds).translate())
print("KRAS prot (len%d):"%(len(prot)-1), prot)
# 4B C-term: ...KMSKDGKKKKKKSKTKCVIM  -> check
print("C-term 20:", prot[-21:-1])
EOF

KRAS prot (len189): MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM*
C-term 20: KISKEEKTPGCVKIKKCIIM

/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python << 'EOF'
from Bio.Seq import Seq
flag_pep="DYKDHDGDYKDHDIDYKDDDDK"
print("3xFLAG pep len:", len(flag_pep))
# Standard 3xFLAG codon-optimized DNA (commonly used)
flag_dna="GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAG"
print("flag dna len", len(flag_dna), "div3", len(flag_dna)%3)
print("translate:", str(Seq(flag_dna).translate()))
print("matches:", str(Seq(flag_dna).translate())==flag_pep)
EOF

3xFLAG pep len: 22
flag dna len 66 div3 0
translate: DYKDHDGDYKDHDIDYKDDDDK
matches: True

/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python << 'EOF'
from Bio import SeqIO
from Bio.Seq import Seq

gb = SeqIO.read("/Users/maxue/Documents/简历/kimi/question/inputs/plvx-egfp-ires-puro.gb","genbank")
vec=str(gb.seq)  # circular, len 8911
kras_t=str(SeqIO.read("/Users/maxue/Documents/简历/kimi/question/inputs/kras_ENST00000256078.fa","fasta").seq)
kras_cds=kras_t[190:190+570]  # ATG...ATGTAA(stop)

flag="GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAG"

# EGFP ORF occupies 2869..3588 (1-based). Python: vec[2868:3588]=ATG..AAGTAA
# We want to REPLACE EGFP ORF with new ORF: ATG+FLAG+KRAS_CDS(incl stop)
# Backbone = everything except 2868:3588.  Linearize backbone by PCR.
# Vector homology arms:
#  - Upstream junction: sequence ending just before EGFP ATG (vec[...:2868]) -> Kozak GCCGCCACC + ATG
#  - Downstream junction: sequence after EGFP stop (vec[3588:...])

# Define vector linear template (rotate so EGFP region is internal). 
# Backbone fragment we PCR: from index 3588 (after stop) forward, wrapping around, to index 2868 (start of ATG).
# Backbone primers: 
#   FWD anneals starting at 3588 forward
#   REV anneals ending at 2868 (reverse of region just upstream of ATG)
up = vec[2868-25:2868]    # 25 bp immediately upstream of EGFP ATG (includes Kozak)
down = vec[3588:3588+25]  # 25 bp immediately downstream of EGFP stop
print("upstream25:", up)
print("downstream25:", down)
print("upstream ends with (Kozak):", vec[2855:2868])
EOF

upstream25: AGGCTCCGCGGCCGCCCCCTTCACC
downstream25: AAGGGTGGGCGCGCCGACCCAGCTT
upstream ends with (Kozak): CGCCCCCTTCACC

/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python << 'EOF'
import asyncio
from Bio import SeqIO
from Bio.Seq import Seq
import labbench2.cloning as c
from labbench2.cloning.sequence_models import BioSequence

gbpath="/Users/maxue/Documents/简历/kimi/question/inputs/plvx-egfp-ires-puro.gb"
fapath="/Users/maxue/Documents/简历/kimi/question/inputs/kras_ENST00000256078.fa"

vec=str(SeqIO.read(gbpath,"genbank").seq)
kras_t=str(SeqIO.read(fapath,"fasta").seq)
kras_cds=kras_t[190:190+570]  # ATG...TAA

flag="GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAG"

def rc(x): return str(Seq(x).reverse_complement())

# --- Backbone PCR (linearize, remove EGFP ORF 2868:3588) ---
# FWD: anneal downstream of EGFP stop, starting at vec[3588:]
bb_fwd_anneal = vec[3588:3588+22]
# REV: anneal upstream of EGFP ATG, ending at vec[:2868]; reverse complement of vec[2868-22:2868]
bb_rev_anneal = rc(vec[2868-22:2868])
bb_fwd = bb_fwd_anneal
bb_rev = bb_rev_anneal
print("bb_fwd", bb_fwd)
print("bb_rev", bb_rev)

# --- Insert PCR: ATG + FLAG + KRAS_CDS ---
# Insert ORF nucleotides we want = ATG + flag + kras_cds (kras_cds already starts ATG and has stop)
# Wait: new ATG then FLAG then KRAS (its own ATG kept). 
insert_orf = "ATG" + flag + kras_cds
print("insert_orf starts", insert_orf[:30])
print("insert prot:", str(Seq(insert_orf).translate())[:40], "...", str(Seq(insert_orf).translate())[-10:])

# Insert FWD primer: upstream homology arm (matches vector upstream incl Kozak) + ATG + FLAG start
# homology arm to vector upstream = vec[2868-25:2868]
up_arm = vec[2868-25:2868]   # 25bp ends ...CTTCACC
# anneal region on template KRAS: but ATG+FLAG aren't in template! So fwd primer = up_arm + ATG + flag + (first ~20nt of kras_cds for annealing)
kras_anneal_fwd = kras_cds[:21]  # KRAS ATGACTGAA...
ins_fwd = up_arm + "ATG" + flag + kras_anneal_fwd
print("\nins_fwd len", len(ins_fwd))
print("ins_fwd", ins_fwd)

# Insert REV primer: downstream homology arm (vector after stop) revcomp + anneal to end of kras_cds
down_arm = vec[3588:3588+25]
kras_anneal_rev = rc(kras_cds[-21:])
ins_rev = rc(down_arm) + kras_anneal_rev
print("\nins_rev len", len(ins_rev))
print("ins_rev", ins_rev)

# Save for next step
import json
json.dump({"bb_fwd":bb_fwd,"bb_rev":bb_rev,"ins_fwd":ins_fwd,"ins_rev":ins_rev},
          open("/tmp/primers.json","w"))
print("saved")
EOF

bb_fwd AAGGGTGGGCGCGCCGACCCAG
bb_rev GGTGAAGGGGGCGGCCGCGGAG
insert_orf starts ATGGACTACAAAGACCATGACGGTGATTAT
insert prot: MDYKDHDGDYKDHDIDYKDDDDKMTEYKLVVVGAGGVGKS ... VKIKKCIIM*

ins_fwd len 115
ins_fwd AGGCTCCGCGGCCGCCCCCTTCACCATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGATGACTGAATATAAACTTGTG

ins_rev len 46
ins_rev AAGCTGGGTCGGCGCGCCCACCCTTTTACATTATAATGCATTTTTT
saved

/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python << 'EOF'
import asyncio, json
from Bio import SeqIO
from Bio.Seq import Seq
import labbench2.cloning as c
from labbench2.cloning.sequence_models import BioSequence

p=json.load(open("/tmp/primers.json"))
gbpath="/Users/maxue/Documents/简历/kimi/question/inputs/plvx-egfp-ires-puro.gb"
fapath="/Users/maxue/Documents/简历/kimi/question/inputs/kras_ENST00000256078.fa"

async def main():
    vecseq = BioSequence.from_file(gbpath)
    krseq  = BioSequence.from_file(fapath)
    print("vec circular?", getattr(vecseq,"is_circular",getattr(vecseq,"circular",None)), "len", len(str(vecseq.sequence)))

    bb = await c.simulate_pcr(vecseq, p["bb_fwd"], p["bb_rev"])
    print("BB product len:", len(str(bb.sequence)))
    ins = await c.simulate_pcr(krseq, p["ins_fwd"], p["ins_rev"])
    print("INS product len:", len(str(ins.sequence)))

    prod = await c.gibson([bb, ins])
    pseq=str(prod.sequence)
    print("GIBSON len:", len(pseq), "circular:", getattr(prod,"is_circular",getattr(prod,"circular",None)))

    # Verify the fusion ORF present
    fusion="ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGATGACTGAA"
    pp=pseq+pseq  # circular wrap
    print("fusion ORF present:", fusion in pp)
    # find ATG..stop reading
    import re
    idx=pp.find(fusion)
    orf=pp[idx:idx+3+66+570]
    print("ORF translate:", str(Seq(orf).translate()))
    # Check IRES & PuroR intact
    print("IRES2/Puro intact check - look for puro start: PuroR vec[4266:4866]")
    vecstr=str(vecseq.sequence)
    puro=vecstr[4266:4866]
    print("PuroR in product:", puro in pp)
    # EGFP should be gone
    egfp=vecstr[2868:3588]
    print("EGFP gone (should be False):", egfp in pp)

asyncio.run(main())
EOF

Exit code 1
vec circular? True len 8911
Traceback (most recent call last):
  File "<stdin>", line 43, in <module>
  File "/opt/homebrew/Cellar/python@3.12/3.12.13_2/Frameworks/Python.framework/Versions/3.12/lib/python3.12/asyncio/runners.py", line 195, in run
    return runner.run(main)
           ^^^^^^^^^^^^^^^^
  File "/opt/homebrew/Cellar/python@3.12/3.12.13_2/Frameworks/Python.framework/Versions/3.12/lib/python3.12/asyncio/runners.py", line 118, in run
    return self._loop.run_until_complete(task)
           ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
  File "/opt/homebrew/Cellar/python@3.12/3.12.13_2/Frameworks/Python.framework/Versions/3.12/lib/python3.12/asyncio/base_events.py", line 691, in run_until_complete
    return future.result()
           ^^^^^^^^^^^^^^^
  File "<stdin>", line 16, in main
TypeError: 'module' object is not callable

/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python << 'EOF'
import labbench2.cloning as c
import inspect
for n in ["simulate_pcr","gibson","enzyme_cut","restriction_assemble","goldengate"]:
    o=getattr(c,n)
    print(n, type(o), "callable" if callable(o) else "NOTcallable")
    if not callable(o):
        print("   members:", [x for x in dir(o) if not x.startswith("_")])
EOF

simulate_pcr <class 'module'> NOTcallable
   members: ['BioSequence', 'LONG_TIMEOUT', 'MINIMUM_AMPLICON_LENGTH', 'Path', 'asyncio', 'convert_fasta', 'json', 'simulate_pcr']
gibson <class 'function'> callable
enzyme_cut <class 'module'> NOTcallable
   members: ['BioSequence', 'RestrictionBatch', 'Seq', 'enzyme_cut']
restriction_assemble <class 'function'> callable
goldengate <class 'function'> callable

/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python << 'EOF'
import asyncio, json
from Bio.Seq import Seq
import labbench2.cloning as c
from labbench2.cloning.sequence_models import BioSequence
sim_pcr = c.simulate_pcr.simulate_pcr
gibson = c.gibson

p=json.load(open("/tmp/primers.json"))
gbpath="/Users/maxue/Documents/简历/kimi/question/inputs/plvx-egfp-ires-puro.gb"
fapath="/Users/maxue/Documents/简历/kimi/question/inputs/kras_ENST00000256078.fa"

async def main():
    vecseq = BioSequence.from_file(gbpath)
    krseq  = BioSequence.from_file(fapath)
    bb = await sim_pcr(vecseq, p["bb_fwd"], p["bb_rev"])
    print("BB len:", len(str(bb.sequence)))
    ins = await sim_pcr(krseq, p["ins_fwd"], p["ins_rev"])
    print("INS len:", len(str(ins.sequence)))
    prod = await gibson([bb, ins])
    pseq=str(prod.sequence)
    print("GIBSON len:", len(pseq), "circ:", getattr(prod,"is_circular",None))
    pp=pseq+pseq
    fusion="ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGATGACTGAA"
    print("fusion present:", fusion in pp)
    idx=pp.find(fusion)
    orf=pp[idx:idx+3+66+570]
    print("ORF prot:", str(Seq(orf).translate()))
    vecstr=str(vecseq.sequence)
    print("PuroR intact:", vecstr[4266:4866] in pp)
    print("EGFP gone:", not (vecstr[2868:3588] in pp))
    # IRES region check: vec around 3588.. (IRES2) — take a chunk after stop
    print("downstream junction intact:", vecstr[3588:3640] in pp)
    # expected size: 8911 - 720(EGFP) + (3+66+570)=639 insert ORF... 
    print("expected len:", 8911-720+ (3+66+570))
asyncio.run(main())
EOF

Exit code 1
BB len: 8191
Traceback (most recent call last):
  File "<stdin>", line 35, in <module>
  File "/opt/homebrew/Cellar/python@3.12/3.12.13_2/Frameworks/Python.framework/Versions/3.12/lib/python3.12/asyncio/runners.py", line 195, in run
    return runner.run(main)
           ^^^^^^^^^^^^^^^^
  File "/opt/homebrew/Cellar/python@3.12/3.12.13_2/Frameworks/Python.framework/Versions/3.12/lib/python3.12/asyncio/runners.py", line 118, in run
    return self._loop.run_until_complete(task)
           ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
  File "/opt/homebrew/Cellar/python@3.12/3.12.13_2/Frameworks/Python.framework/Versions/3.12/lib/python3.12/asyncio/base_events.py", line 691, in run_until_complete
    return future.result()
           ^^^^^^^^^^^^^^^
  File "<stdin>", line 17, in main
  File "/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/src/labbench2/cloning/simulate_pcr.py", line 83, in simulate_pcr
    raise ValueError("PCR simulation ran successfully, but no amplicon was observed.")
ValueError: PCR simulation ran successfully, but no amplicon was observed.

/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python << 'EOF'
import asyncio, json
from Bio.Seq import Seq
import labbench2.cloning as c
from labbench2.cloning.sequence_models import BioSequence
sim_pcr=c.simulate_pcr.simulate_pcr
p=json.load(open("/tmp/primers.json"))
fapath="/Users/maxue/Documents/简历/kimi/question/inputs/kras_ENST00000256078.fa"

async def main():
    kr=BioSequence.from_file(fapath)
    krs=str(kr.sequence)
    # anneal portions
    fa=p["ins_fwd"][-21:]; ra=p["ins_rev"][-21:]
    print("fwd anneal:", fa, "in template:", fa in krs)
    print("rev anneal rc in template:", str(Seq(ra).reverse_complement()) in krs)
    # try shorter rev anneal & check primer-length constraints
    for revtail in [21,25,30]:
        try:
            ins=await sim_pcr(kr, p["ins_fwd"], p["ins_rev"][:-21]+p["ins_rev"][-revtail:] if False else p["ins_rev"])
            print("ok", len(str(ins.sequence)))
            break
        except Exception as e:
            print("fail", e)
            break
    # Try with plain anneal-only primers (no tails) to confirm PCR works
    try:
        ins=await sim_pcr(kr, p["ins_fwd"][-21:], p["ins_rev"][-21:])
        print("anneal-only product len:", len(str(ins.sequence)))
    except Exception as e:
        print("anneal-only fail:", e)
asyncio.run(main())
EOF

fwd anneal: ATGACTGAATATAAACTTGTG in template: True
rev anneal rc in template: True
fail PCR simulation ran successfully, but no amplicon was observed.
anneal-only fail: PCR simulation ran successfully, but no amplicon was observed.

/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python -c "
import labbench2.cloning as c, inspect
print(inspect.getsource(c.simulate_pcr.simulate_pcr))
" 2>/dev/null | head -90

async def simulate_pcr(
    sequence: BioSequence,
    forward_primer: BioSequence | str,
    reverse_primer: BioSequence | str,
) -> BioSequence:
    """Simulate PCR and return the amplicon sequence.

    The Go binary is automatically compiled for your OS/architecture on first use.
    Requires Go 1.21+ to be installed.
    """
    primers_binary = _get_primers_binary()

    fasta_str, circular = convert_fasta(sequence.to_fasta())
    seq_only = "".join(fasta_str.splitlines()[1:])

    command = [
        str(primers_binary),
        "-sequence",
        seq_only,
        "-forward-overhang",
        "",
        "-reverse-overhang",
        "",
        "-target-tm",
        "45.0",
        "-forward-primer",
        _get_seq(forward_primer),
        "-reverse-primer",
        _get_seq(reverse_primer),
    ]
    if circular:
        command.append("-circular")

    proc = await asyncio.create_subprocess_exec(
        *command, stdout=asyncio.subprocess.PIPE, stderr=asyncio.subprocess.PIPE
    )

    try:
        stdout, stderr = await asyncio.wait_for(proc.communicate(), timeout=LONG_TIMEOUT)
    except TimeoutError as e:
        proc.kill()
        await proc.communicate()
        raise RuntimeError("Simulating PCR timed out") from e

    if proc.returncode != 0:
        raise RuntimeError(f"Subprocess failed with error: {stderr.decode()}")

    try:
        data = json.loads(stdout.decode())
        amplicon_fasta = data["amplicon_fasta"]
    except Exception as e:
        raise RuntimeError("Failed to simulate PCR for these inputs.") from e

    if len(amplicon_fasta) < MINIMUM_AMPLICON_LENGTH:
        raise ValueError("PCR simulation ran successfully, but no amplicon was observed.")

    return BioSequence.from_fasta(amplicon_fasta, is_content=True)

/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python << 'EOF'
import asyncio
from Bio.Seq import Seq
from Bio.SeqUtils import MeltingTemp as mt
import labbench2.cloning as c
from labbench2.cloning.sequence_models import BioSequence
print("MIN_AMPLICON:", c.simulate_pcr.MINIMUM_AMPLICON_LENGTH)
sim=c.simulate_pcr.simulate_pcr
fapath="/Users/maxue/Documents/简历/kimi/question/inputs/kras_ENST00000256078.fa"

from Bio import SeqIO
krs=str(SeqIO.read(fapath,"fasta").seq)
cds=krs[190:190+570]
def rc(x): return str(Seq(x).reverse_complement())
async def main():
    kr=BioSequence.from_file(fapath)
    # try different anneal lengths for fwd & rev
    for L in [21,25,30,35]:
        fa=cds[:L]; ra=rc(cds[-L:])
        print(f"L={L} fwdTm={mt.Tm_NN(Seq(fa)):.1f} revTm={mt.Tm_NN(Seq(ra)):.1f}")
        try:
            x=await sim(kr,fa,ra); print("   amplicon", len(str(x.sequence)))
        except Exception as e: print("   fail:", str(e)[:50])
asyncio.run(main())
EOF

MIN_AMPLICON: 7
L=21 fwdTm=43.5 revTm=39.8
   fail: PCR simulation ran successfully, but no amplicon w
L=25 fwdTm=49.0 revTm=43.0
   amplicon 570
L=30 fwdTm=54.8 revTm=48.5
   amplicon 570
L=35 fwdTm=60.1 revTm=54.4
   amplicon 570

/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python << 'EOF'
import asyncio, json
from Bio.Seq import Seq
from Bio import SeqIO
import labbench2.cloning as c
from labbench2.cloning.sequence_models import BioSequence
sim=c.simulate_pcr.simulate_pcr; gibson=c.gibson

gbpath="/Users/maxue/Documents/简历/kimi/question/inputs/plvx-egfp-ires-puro.gb"
fapath="/Users/maxue/Documents/简历/kimi/question/inputs/kras_ENST00000256078.fa"
vec=str(SeqIO.read(gbpath,"genbank").seq)
krs=str(SeqIO.read(fapath,"fasta").seq)
cds=krs[190:190+570]
flag="GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAG"
def rc(x): return str(Seq(x).reverse_complement())

# Backbone primers (already validated)
bb_fwd=vec[3588:3588+22]
bb_rev=rc(vec[2868-22:2868])

# Insert primers: 25nt anneal + homology arms (>=25bp to vector)
up_arm=vec[2868-25:2868]          # upstream homology incl Kozak ...CTTCACC
down_arm=vec[3588:3588+25]        # downstream homology
ins_fwd=up_arm+"ATG"+flag+cds[:25]
ins_rev=rc(down_arm)+rc(cds[-25:])
print("ins_fwd",len(ins_fwd),ins_fwd)
print("ins_rev",len(ins_rev),ins_rev)

async def main():
    vecseq=BioSequence.from_file(gbpath); kr=BioSequence.from_file(fapath)
    bb=await sim(vecseq,bb_fwd,bb_rev); print("BB",len(str(bb.sequence)))
    ins=await sim(kr,ins_fwd,ins_rev);  print("INS",len(str(ins.sequence)))
    prod=await gibson([bb,ins]); pseq=str(prod.sequence)
    print("GIBSON len",len(pseq),"circ",getattr(prod,"is_circular",None))
    pp=pseq+pseq
    fusion="ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGATGACTGAA"
    print("fusion present:",fusion in pp)
    idx=pp.find(fusion); orf=pp[idx:idx+3+66+570]
    prot=str(Seq(orf).translate())
    print("ORF prot:",prot)
    print("single stop, correct end:", prot.count("*")==1 and prot.endswith("CIIM*"))
    print("PuroR intact:",vec[4266:4866] in pp)
    print("EGFP gone:",not(vec[2868:3588] in pp))
    print("Kozak+ATG context:", pp[idx-9:idx+3])
    print("expected len 8911-720+639=",8911-720+639)
    json.dump({"bb_fwd":bb_fwd,"bb_rev":bb_rev,"ins_fwd":ins_fwd,"ins_rev":ins_rev},open("/tmp/primers.json","w"))
asyncio.run(main())
EOF

Exit code 1
ins_fwd 119 AGGCTCCGCGGCCGCCCCCTTCACCATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGATGACTGAATATAAACTTGTGGTAG
ins_rev 50 AAGCTGGGTCGGCGCGCCCACCCTTTTACATTATAATGCATTTTTTAATT
BB 8191
INS 689
Traceback (most recent call last):
  File "<stdin>", line 46, in <module>
  File "/opt/homebrew/Cellar/python@3.12/3.12.13_2/Frameworks/Python.framework/Versions/3.12/lib/python3.12/asyncio/runners.py", line 195, in run
    return runner.run(main)
           ^^^^^^^^^^^^^^^^
  File "/opt/homebrew/Cellar/python@3.12/3.12.13_2/Frameworks/Python.framework/Versions/3.12/lib/python3.12/asyncio/runners.py", line 118, in run
    return self._loop.run_until_complete(task)
           ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
  File "/opt/homebrew/Cellar/python@3.12/3.12.13_2/Frameworks/Python.framework/Versions/3.12/lib/python3.12/asyncio/base_events.py", line 691, in run_until_complete
    return future.result()
           ^^^^^^^^^^^^^^^
  File "<stdin>", line 32, in main
TypeError: object list can't be used in 'await' expression

/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python -c "
import labbench2.cloning as c, inspect
print('iscoroutine:', inspect.iscoroutinefunction(c.gibson))
print(inspect.signature(c.gibson))
src=inspect.getsource(c.gibson)
print(src[:1500])
"

iscoroutine: False
(sequences: list[labbench2.cloning.sequence_models.BioSequence], min_overlap: int = 10, max_overlap: int = 60) -> list[labbench2.cloning.sequence_models.BioSequence]
def gibson(
    sequences: list[BioSequence], min_overlap: int = 10, max_overlap: int = 60
) -> list[BioSequence]:
    """Predict Gibson Assembly outcome from DNA sequences."""
    fragments = sequences[:]
    circulars: list[BioSequence] = []
    linears: dict[str, BioSequence] = {}

    def assemble(current: BioSequence, used: set[str]):
        extended = False
        for frag in fragments:
            if frag.name in used:
                continue

            for suffix, frag_seq in [
                ("", frag.sequence),
                ("-rev", reverse_complement(frag.sequence)),
            ]:
                overlap = find_homology_length(current.sequence, frag_seq, min_overlap, max_overlap)
                if overlap >= min_overlap:
                    extended = True
                    new_seq = BioSequence(
                        sequence=current.sequence + frag_seq[overlap:],
                        is_circular=False,
                        name=make_pretty_id(f"{current.name}-{frag.name}{suffix}"),
                    )
                    assemble(new_seq, used | {frag.name})

        if not extended:
            self_overlap = find_self_homology_length(current.sequence, min_overlap, max_overlap)
            is_circ = self_overlap >= min_overlap
            seq = current.sequence[:-self_overlap] if is_circ else current.sequence

            result = BioSequence(
                sequence=seq,
                is_circular=is_circ,
                name=make_pret

/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python << 'EOF'
import asyncio, json
from Bio.Seq import Seq
from Bio import SeqIO
import labbench2.cloning as c
from labbench2.cloning.sequence_models import BioSequence
sim=c.simulate_pcr.simulate_pcr; gibson=c.gibson
p=json.load(open("/tmp/primers.json"))
gbpath="/Users/maxue/Documents/简历/kimi/question/inputs/plvx-egfp-ires-puro.gb"
fapath="/Users/maxue/Documents/简历/kimi/question/inputs/kras_ENST00000256078.fa"
vec=str(SeqIO.read(gbpath,"genbank").seq)

async def main():
    vecseq=BioSequence.from_file(gbpath); kr=BioSequence.from_file(fapath)
    bb=await sim(vecseq,p["bb_fwd"],p["bb_rev"])
    ins=await sim(kr,p["ins_fwd"],p["ins_rev"])
    print("BB",len(str(bb.sequence)),"INS",len(str(ins.sequence)))
    prods=gibson([bb,ins])
    print("num products:",len(prods))
    for pr in prods:
        print("  product len",len(str(pr.sequence)),"circ",pr.is_circular,"name",pr.name)
    # pick circular product
    circ=[pr for pr in prods if pr.is_circular]
    prod=circ[0] if circ else prods[0]
    pseq=str(prod.sequence); pp=pseq+pseq
    fusion="ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGATGACTGAA"
    print("circular len",len(pseq))
    print("fusion present:",fusion in pp)
    idx=pp.find(fusion); orf=pp[idx:idx+3+66+570]
    prot=str(Seq(orf).translate())
    print("ORF prot:",prot)
    print("end CIIM*:",prot.endswith("CIIM*"),"single stop:",prot.count("*")==1)
    print("3xFLAG immediately after init Met:",prot[1:23]=="DYKDHDGDYKDHDIDYKDDDDK")
    print("PuroR intact:",vec[4266:4866] in pp)
    print("EGFP gone:",not(vec[2868:3588] in pp))
    print("expected len:",8911-720+639)
asyncio.run(main())
EOF

Exit code 1
Traceback (most recent call last):
  File "<stdin>", line 36, in <module>
  File "/opt/homebrew/Cellar/python@3.12/3.12.13_2/Frameworks/Python.framework/Versions/3.12/lib/python3.12/asyncio/runners.py", line 195, in run
    return runner.run(main)
           ^^^^^^^^^^^^^^^^
  File "/opt/homebrew/Cellar/python@3.12/3.12.13_2/Frameworks/Python.framework/Versions/3.12/lib/python3.12/asyncio/runners.py", line 118, in run
    return self._loop.run_until_complete(task)
           ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
  File "/opt/homebrew/Cellar/python@3.12/3.12.13_2/Frameworks/Python.framework/Versions/3.12/lib/python3.12/asyncio/base_events.py", line 691, in run_until_complete
    return future.result()
           ^^^^^^^^^^^^^^^
  File "<stdin>", line 15, in main
  File "/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/src/labbench2/cloning/simulate_pcr.py", line 83, in simulate_pcr
    raise ValueError("PCR simulation ran successfully, but no amplicon was observed.")
ValueError: PCR simulation ran successfully, but no amplicon was observed.

/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python << 'EOF'
import asyncio, json
from Bio.Seq import Seq
from Bio import SeqIO
import labbench2.cloning as c
from labbench2.cloning.sequence_models import BioSequence
sim=c.simulate_pcr.simulate_pcr; gibson=c.gibson
gbpath="/Users/maxue/Documents/简历/kimi/question/inputs/plvx-egfp-ires-puro.gb"
fapath="/Users/maxue/Documents/简历/kimi/question/inputs/kras_ENST00000256078.fa"
vec=str(SeqIO.read(gbpath,"genbank").seq)
krs=str(SeqIO.read(fapath,"fasta").seq); cds=krs[190:190+570]
flag="GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAG"
def rc(x): return str(Seq(x).reverse_complement())

bb_fwd=vec[3588:3588+22]
bb_rev=rc(vec[2868-22:2868])
up_arm=vec[2868-25:2868]; down_arm=vec[3588:3588+25]
ins_fwd=up_arm+"ATG"+flag+cds[:25]
ins_rev=rc(down_arm)+rc(cds[-25:])

async def main():
    vecseq=BioSequence.from_file(gbpath); kr=BioSequence.from_file(fapath)
    bb=await sim(vecseq,bb_fwd,bb_rev)
    ins=await sim(kr,ins_fwd,ins_rev)
    print("BB",len(str(bb.sequence)),"INS",len(str(ins.sequence)))
    prods=gibson([bb,ins])
    circ=[pr for pr in prods if pr.is_circular]
    print("products:",[(len(str(pr.sequence)),pr.is_circular) for pr in prods])
    prod=circ[0]; pseq=str(prod.sequence); pp=pseq+pseq
    fusion="ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGATGACTGAA"
    idx=pp.find(fusion); orf=pp[idx:idx+639]; prot=str(Seq(orf).translate())
    print("circular len",len(pseq),"(expect",8911-720+639,")")
    print("fusion present:",fusion in pp)
    print("ORF prot:",prot)
    print("FLAG after init Met:",prot[1:23]=="DYKDHDGDYKDHDIDYKDDDDK","| end CIIM*:",prot.endswith("CIIM*"),"| 1 stop:",prot.count("*")==1)
    print("PuroR intact:",vec[4266:4866] in pp,"| EGFP gone:",not(vec[2868:3588] in pp))
    json.dump({"bb_fwd":bb_fwd,"bb_rev":bb_rev,"ins_fwd":ins_fwd,"ins_rev":ins_rev},open("/tmp/primers.json","w"))
    print("PRIMERS SAVED")
asyncio.run(main())
EOF

BB 8191 INS 689
products: [(8830, True)]
circular len 8830 (expect 8830 )
fusion present: True
ORF prot: MDYKDHDGDYKDHDIDYKDDDDKMTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM*
FLAG after init Met: True | end CIIM*: True | 1 stop: True
PuroR intact: True | EGFP gone: True
PRIMERS SAVED

/Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python -c "
import labbench2.cloning as c, inspect
print(inspect.signature(c.CloningProtocol.from_file))
print('---run---')
print(inspect.signature(c.CloningProtocol.run))
print('iscoro run:', inspect.iscoroutinefunction(c.CloningProtocol.run))
"

(file_path: 'Path | str', tag_open: 'str' = '<protocol>', tag_close: 'str' = '</protocol>') -> 'CloningProtocol'
---run---
(self, base_dir: 'Path | str') -> 'list[BioSequence]'
iscoro run: True

cat > /tmp/test_protocol.txt << 'PROTO'
<protocol>
gibson(
  pcr(plvx-egfp-ires-puro.gb, "AAGGGTGGGCGCGCCGACCCAG", "GGTGAAGGGGGCGGCCGCGGAG"),
  pcr(kras_ENST00000256078.fa, "AGGCTCCGCGGCCGCCCCCTTCACCATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGATGACTGAATATAAACTTGTGGTAG", "AAGCTGGGTCGGCGCGCCCACCCTTTTACATTATAATGCATTTTTTAATT")
)
</protocol>
PROTO
# Need input files reachable by base name. Copy inputs to a working dir.
mkdir -p /tmp/protorun && cp /Users/maxue/Documents/简历/kimi/question/inputs/plvx-egfp-ires-puro.gb /Users/maxue/Documents/简历/kimi/question/inputs/kras_ENST00000256078.fa /tmp/protorun/
cp /tmp/test_protocol.txt /tmp/protorun/
echo done

done

cd /tmp/protorun && /Users/maxue/Documents/简历/deepseek/bio/external/labbench2/.venv/bin/python << 'EOF'
import asyncio
from Bio.Seq import Seq
import labbench2.cloning as c
async def main():
    proto=c.CloningProtocol.from_file("/tmp/protorun/test_protocol.txt")
    res=await proto.run(base_dir="/tmp/protorun")
    print("results:",len(res))
    for r in res:
        s=str(r.sequence)
        print("len",len(s),"circ",r.is_circular)
        pp=s+s
        fusion="ATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGATGACTGAA"
        idx=pp.find(fusion)
        if idx>=0:
            prot=str(Seq(pp[idx:idx+639]).translate())
            print("  fusion OK, prot end:",prot[-12:],"len(aa)",len(prot)-1)
asyncio.run(main())
EOF

results: 1
len 8830 circ True
  fusion OK, prot end: GCVKIKKCIIM* len(aa) 212

{"file_path": "/Users/maxue/Documents/简历/kimi/results/agent_answer.txt", "content": "<protocol>\ngibson(\n  pcr(plvx-egfp-ires-puro.gb, \"AAGGGTGGGCGCGCCGACCCAG\", \"GGTGAAGGGGGCGGCCGCGGAG\"),\n  pcr(kras_ENST00000256078.fa, \"AGGCTCCGCGGCCGCCCCCTTCACCATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGATGACTGAATATAAACTTGTGGTAG\", \"AAGCTGGGTCGGCGCGCCCACCCTTTTACATTATAATGCATTTTTTAATT\")\n)\n</protocol>\n"}

File created successfully at: /Users/maxue/Documents/简历/kimi/results/agent_answer.txt (file state is current in your context — no need to Read it back)