pGEM-3Z plasmid map

How it is computed

• Feature annotation from the library. Local alignment with affine gaps, seeded by shared 12-base words and banded; match +2, mismatch -3, this app's parameters, checked against the Python reference (Biopython 1.88's PairwiseAligner in local mode); a gap of k bases scores -5 - 2(k - 1), so one long gap costs less than several short ones. Identity is identical columns over alignment columns, gaps included; coverage is the feature's aligned bases over its length. The exported local_alignment_score is this score, for re-deriving a call, not a quality measure; for a feature under 30 bases no alignment runs and the score is set to twice its length, the score a whole exact alignment would have. A coding feature the library finds carries the /transl_table of the record it was taken from where that record names a genetic code other than genetic code 1, and carries none otherwise. Attribution to the original publications not yet checked.Checked against Biopython 1.88 PairwiseAligner with the same scoring on 26 alignments of library parts to other NCBI records: identity and coverage within 0.1 percentage points, place, strand, call and score exact; 12 synthetic inserts on each side of every threshold; no match and 0 partial calls on 10 random sequences of 10,000 bp and both golden plasmids reversed (2026-10-06). Library 2 (2026-10-17): all 4,005 pairs of its parts aligned against each other with the same scoring, 66 of them reaching the thresholds and each published with how it is shown; no call on 12 random controls; what is shown at a locus, the parts named beside it and the inside labels exact on 14 sequences.
• Restriction sites and cut positions. Recognition sequences and cut positions as published facts, typed by hand (pipeline/plasmid-map/enzymes.csv); every row checked against Biopython 1.88 Bio.Restriction, whose data are REBASE release 404 (Roberts RJ et al. REBASE: a database for DNA restriction and modification. Nucleic Acids Res 2023;51:D629-D630).Every row checked against Biopython 1.88 Bio.Restriction (enzyme data release 404) in its own notation, overhang and cut positions: 223 enzymes on pUC19 and pBR322, 446 cases, cut positions exact, with sites across the origin and 6 double digests (2026-10-06).
• dam and dcm methylation of sites. McClelland M, Nelson M, Raschke E. Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases. Nucleic Acids Res 1994;22:3640-3659, Table I.Checked against 19 cases worked by hand from McClelland et al. 1994, Table I (one per outcome, flank and origin overlaps, fully methylated DpnI and host settings, plus hemi-site sensitivity entries for other enzymes; not a hemimethylated-DpnI model), and the cited blocked examples on pUC19 (MboI) and pBR322 (EcoRII), outcomes exact (2026-10-06). Which enzymes need a Table I row (82 whose site can carry a dam or dcm methylated base, 57 of them with a row) checked by a second implementation sharing no code with the engine (2026-10-17); whether Table I lists the other 25 has not been checked.
• Sequencing-primer sites. Exact and one-mismatch matches of the shipped primers on both strands; a mismatch is reported with its distance from the primer's 3′ end and its own position, and nothing is claimed about whether such a site primes. A template letter other than A, C, G or T is an unknown base, not a mismatch: a site may hold one in place of the mismatch, and is then not exact. An oligo with more than 200 sites has none drawn or listed.Each of the 6 shipped primers found exactly at its NCBI binding site at build and every week; one-mismatch places unit-tested (2026-10-06).
• ng, pmol, copies and ligation amounts. pmol = ng × 1000 / MW; copies = ng × 10⁻⁹ × N_A / MW, with N_A = 6.02214076 × 10²³ per mole (exact, SI 2019); insert ng = vector ng × insert bp / vector bp × ratio. MW of the double-stranded plasmid from its own sequence (both strands, no end groups when circular) with the CIAAW 2021 atomic weights.Checked against exact rational arithmetic on 11 cases (pmol, copies per µL, ng for copies, ligation, the 650 Da per bp rule and the weights of pUC19 and pBR322), to full double precision (2026-10-06).

Limits

• For research and education only, and not medical advice: this app designs restriction, Golden Gate and Gibson products from the sequences you supply; a designed product is not evidence that the reaction occurred, and nothing here predicts whether a reaction works, how well, or whether the product is expressed.
• Enzyme sites and cut positions are from a hand-curated table of 223 enzymes (version 1, built 2026-10-06); it may differ from a vendor's list, so check NEBcutter before a critical digest. Star activity, Type IIB and nicking enzymes are not modelled.
• Methylation blocking is called only where McClelland et al. (1994) record it; elsewhere a site reads 'may be blocked: sensitivity not recorded', which means unknown, never 'cuts'. 25 of the 82 enzymes whose site can carry a dam or dcm methylated base have no row in this app's copy of Table I, and whether Table I lists them has not been checked.
• Feature badges are alignment matches to public records: evidence of sequence, not of function.
• Primer sites are exact and one-mismatch sites of the shipped primers; whether a primer primes at a mismatched site is not assessed, and a Tm is given only against a perfect match.

How these numbers were checked