CALCULATE WITH CONFIDENCE

Restriction Cloning & Plasmids

Golden Gate Ordered Junction Audit Workbench

Retain an entered linear or circular fragment order, verify its junction ledger, and flag literal duplicate, reverse-complement-reused, palindromic and extreme-composition 3- or 4-base overhangs without inventing a fidelity score.

Biology · experimental measurements

Turn a hand-written Type IIS assembly order into an auditable junction graph while leaving sequence design, empirical fidelity, enzyme choice and protocol approval to validated resources.

Private calculations in your browser · explicit inputs and model boundaries
Example preview · Linear four-partEntered junction overhang composition
Vector left→Promoter · AATG1 GC bases
Promoter→CDS · GCTT2 GC bases
CDS→Vector right · CGAC3 GC bases

Each bar shows the literal GC-base count of one ordered product-strand overhang. Composition alone is not an assembly-fidelity estimate.

  1. 1EnterProvide the known values
  2. 2CalculateResults update automatically
  3. 3VerifyReview the details and units
Try an example

Retained fragments: label, retained length bp
1 row
Row 1

Empty rows are ignored until edited. Keep commas and tabs out of individual entries; use the paste view for comma- or tab-separated records.

Junctions: upstream label, downstream label, product-strand overhang 5′→3′
1 row
Row 1

Empty rows are ignored until edited. Keep commas and tabs out of individual entries; use the paste view for comma- or tab-separated records.

Calculation result

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Understand the relationship

The reasoning behind the result

Topology determines the expected graph

linear: n−1 junctions; circular: n junctions

Every internal adjacent pair needs one entered junction.

A circular plan also closes the last fragment back to the first.

Overhang relationships are literal observations

palindrome when h = reverse-complement(h)

Exact reuse, reverse-complement reuse and palindromy can be detected from the entered strings.

These labels are not probabilities or universal failure rules.

Fidelity is empirical and system-specific

Assembly behavior depends on the enzyme, ligase, overhang set, reaction condition and neighboring sequence context.

A complete design must also verify recognition-site orientation, domestication, frame, scar and retained sequence.

Follow the numbers

Audit a circular three-fragment plan

  1. Retain the entered fragment order Backbone→Insert A→Insert B.
  2. Expect three junctions because the construct is circular.
  3. Match each entered upstream–downstream pair to that order.
  4. Compare the three product-strand overhangs for exact, reverse-complement and palindrome relationships.

The ledger verifies the declared order and flags literal sequence relationships without predicting assembly fidelity.

Quick guide

How to use this calculator

  1. List fragments in intended construct order using retained product lengths.
  2. List every adjacent ordered junction using one consistent product-strand 5′→3′ convention.
  3. Resolve flagged observations with enzyme-specific, experimentally measured overhang-fidelity data and the complete sequence design.

Calculation method

Calculation and interpretation

Turn a hand-written Type IIS assembly order into an auditable junction graph while leaving sequence design, empirical fidelity, enzyme choice and protocol approval to validated resources.

Expected ordered junctions are fragment i→i+1, plus last→first for a circular construct. Overhang flags compare exact sequences, reverse complements, palindromy and GC count.

Worked example

Audit a circular three-fragment plan

The ledger verifies the declared order and flags literal sequence relationships without predicting assembly fidelity.

Expected ordered junctions are fragment i→i+1, plus last→first for a circular construct. Overhang flags compare exact sequences, reverse complements, palindromy and GC count.

Supported inputs

Precision and limits

Entered graph only

The workbench does not parse full DNA sequences or discover Type IIS recognition and cut sites.

One orientation convention

Every overhang must use the same visitor-declared product-strand 5′→3′ convention.

No fidelity score

No empirical ligation matrix, probability, pass/fail threshold or preferred overhang is supplied.

No protocol selection

Enzyme, ligase, temperature cycles, concentrations and cleanup remain external.

Retained lengths

Construct length is the sum of entered retained fragment lengths; added or removed scars must already be represented.

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