From e96a63e419525529b8408bbac5d433ab55aed18b Mon Sep 17 00:00:00 2001 From: Trenton w Fleming Date: Wed, 15 Nov 2023 00:09:47 -0500 Subject: [PATCH] Cloning refactor and data race fix (#393) * enzyme manager to avoid managing global state over the lifetime of a program * clone_test -> clone to make it easier to test and bench * remove concurrency * remove todos * expose some enzymes * fix lint * update changelog and remove pointer reciever. * moved example test to separate file for namespace clarity in rendered go doc examples. * renamed variables and added comments. * renamed variables and removed unnecessary control block. * added struct field name to changelog. * Golden Gate is no longer a receiver * update changelog --------- Co-authored-by: Timothy Stiles --- CHANGELOG.md | 9 +- clone/clone.go | 252 +++++++++++++++++++++--------------------- clone/clone_test.go | 173 +++++++++++++++-------------- clone/example_test.go | 31 ++++++ 4 files changed, 255 insertions(+), 210 deletions(-) create mode 100644 clone/example_test.go diff --git a/CHANGELOG.md b/CHANGELOG.md index c2fe0c054..fc655a687 100644 --- a/CHANGELOG.md +++ b/CHANGELOG.md @@ -14,9 +14,16 @@ and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0 ### Fixed - `fastq` parser no longer becomes de-aligned when reading (#325) - `fastq` now handles optionals correctly (#323) +- No more data race in GoldenGate (#276) + +### Breaking +- CutWithEnzymeByName is now a receiver of EnzymeManager. GoldenGate now takes an Enzyme instead of the name of an enzyme. +This is an effort to remove dependence on some package level global state and build some flexibility managing enzymes +over the lifetime of the program. +- Enzyme.OverhangLen is now named Enzyme.OverhangLength ## [0.26.0] - 2023-07-22 Oops, we weren't keeping a changelog before this tag! [unreleased]: https://github.com/TimothyStiles/poly/compare/v0.26.0...main -[0.26.0]: https://github.com/TimothyStiles/poly/releases/tag/v0.26.0 \ No newline at end of file +[0.26.0]: https://github.com/TimothyStiles/poly/releases/tag/v0.26.0 diff --git a/clone/clone.go b/clone/clone.go index e2cbf41ce..cc9939ba1 100644 --- a/clone/clone.go +++ b/clone/clone.go @@ -46,7 +46,6 @@ import ( "regexp" "sort" "strings" - "sync" "github.com/TimothyStiles/poly/checks" "github.com/TimothyStiles/poly/seqhash" @@ -83,15 +82,27 @@ type Enzyme struct { RegexpFor *regexp.Regexp RegexpRev *regexp.Regexp Skip int - OverhangLen int + OverheadLength int RecognitionSite string } -// Eventually, we want to get the data for this map from ftp://ftp.neb.com/pub/rebase -var enzymeMap = map[string]Enzyme{ - "BsaI": {"BsaI", regexp.MustCompile("GGTCTC"), regexp.MustCompile("GAGACC"), 1, 4, "GGTCTC"}, - "BbsI": {"BbsI", regexp.MustCompile("GAAGAC"), regexp.MustCompile("GTCTTC"), 2, 4, "GAAGAC"}, - "BtgZI": {"BtgZI", regexp.MustCompile("GCGATG"), regexp.MustCompile("CATCGC"), 10, 4, "GCGATG"}, +// EnzymeManager manager for Enzymes. Allows for management of enzymes throughout the lifecyle of your +// program. EnzymeManager is not safe for concurrent use. +type EnzymeManager struct { + // enzymeMap Map of enzymes that exist for the lifetime of the manager. Not safe for concurrent use. + enzymeMap map[string]Enzyme +} + +// NewEnzymeManager creates a new EnzymeManager given some enzymes. +func NewEnzymeManager(enzymes []Enzyme) EnzymeManager { + enzymeMap := make(map[string]Enzyme) + for enzymeIndex := range enzymes { + enzymeMap[enzymes[enzymeIndex].Name] = enzymes[enzymeIndex] + } + + return EnzymeManager{ + enzymeMap: enzymeMap, + } } /****************************************************************************** @@ -100,30 +111,37 @@ Base cloning functions begin here. ******************************************************************************/ -func getBaseRestrictionEnzymes() map[string]Enzyme { - return enzymeMap -} - // CutWithEnzymeByName cuts a given sequence with an enzyme represented by the // enzyme's name. It is a convenience wrapper around CutWithEnzyme that // allows us to specify the enzyme by name. -func CutWithEnzymeByName(seq Part, directional bool, enzymeStr string) ([]Fragment, error) { - enzymeMap := getBaseRestrictionEnzymes() - if _, ok := enzymeMap[enzymeStr]; !ok { - return []Fragment{}, errors.New("Enzyme " + enzymeStr + " not found in enzymeMap") +func (enzymeManager EnzymeManager) CutWithEnzymeByName(part Part, directional bool, name string) ([]Fragment, error) { + // Get the enzyme from the enzyme map + enzyme, err := enzymeManager.GetEnzymeByName(name) + if err != nil { + // Return an error if there was an error + return []Fragment{}, err } - enzyme := enzymeMap[enzymeStr] - return CutWithEnzyme(seq, directional, enzyme), nil + // Cut the sequence with the enzyme + return CutWithEnzyme(part, directional, enzyme), nil +} + +// GetEnzymeByName gets the enzyme by it's name. If the enzyme manager does not +// contain an enzyme with the provided name, an error will be returned +func (enzymeManager EnzymeManager) GetEnzymeByName(name string) (Enzyme, error) { + if enzyme, ok := enzymeManager.enzymeMap[name]; ok { + return enzyme, nil + } + return Enzyme{}, errors.New("Enzyme " + name + " not found") } // CutWithEnzyme cuts a given sequence with an enzyme represented by an Enzyme struct. -func CutWithEnzyme(seq Part, directional bool, enzyme Enzyme) []Fragment { - var fragmentSeqs []string +func CutWithEnzyme(part Part, directional bool, enzyme Enzyme) []Fragment { + var fragmentSequences []string var sequence string - if seq.Circular { - sequence = strings.ToUpper(seq.Sequence + seq.Sequence) + if part.Circular { + sequence = strings.ToUpper(part.Sequence + part.Sequence) } else { - sequence = strings.ToUpper(seq.Sequence) + sequence = strings.ToUpper(part.Sequence) } // Check for palindromes @@ -135,20 +153,20 @@ func CutWithEnzyme(seq Part, directional bool, enzyme Enzyme) []Fragment { var reverseOverhangs []Overhang forwardCuts := enzyme.RegexpFor.FindAllStringIndex(sequence, -1) for _, forwardCut := range forwardCuts { - forwardOverhangs = append(forwardOverhangs, Overhang{Length: enzyme.OverhangLen, Position: forwardCut[1] + enzyme.Skip, Forward: true, RecognitionSitePlusSkipLength: len(enzyme.RecognitionSite) + enzyme.Skip}) + forwardOverhangs = append(forwardOverhangs, Overhang{Length: enzyme.OverheadLength, Position: forwardCut[1] + enzyme.Skip, Forward: true, RecognitionSitePlusSkipLength: len(enzyme.RecognitionSite) + enzyme.Skip}) } // Palindromic enzymes won't need reverseCuts if !palindromic { reverseCuts := enzyme.RegexpRev.FindAllStringIndex(sequence, -1) for _, reverseCut := range reverseCuts { - reverseOverhangs = append(reverseOverhangs, Overhang{Length: enzyme.OverhangLen, Position: reverseCut[0] - enzyme.Skip, Forward: false, RecognitionSitePlusSkipLength: len(enzyme.RecognitionSite) + enzyme.Skip}) + reverseOverhangs = append(reverseOverhangs, Overhang{Length: enzyme.OverheadLength, Position: reverseCut[0] - enzyme.Skip, Forward: false, RecognitionSitePlusSkipLength: len(enzyme.RecognitionSite) + enzyme.Skip}) } } - // If, on a linear sequence, the last overhang's position + EnzymeSkip + EnzymeOverhangLen is over the length of the sequence, remove that overhang. + // If, on a linear sequence, the last overhang's position + EnzymeSkip + EnzymeOverhangLength is over the length of the sequence, remove that overhang. for _, overhangSet := range [][]Overhang{forwardOverhangs, reverseOverhangs} { if len(overhangSet) > 0 { - if !seq.Circular && (overhangSet[len(overhangSet)-1].Position+enzyme.Skip+enzyme.OverhangLen > len(sequence)) { + if !part.Circular && (overhangSet[len(overhangSet)-1].Position+enzyme.Skip+enzyme.OverheadLength > len(sequence)) { overhangSet = overhangSet[:len(overhangSet)-1] } } @@ -166,26 +184,26 @@ func CutWithEnzyme(seq Part, directional bool, enzyme Enzyme) []Fragment { var nextOverhang Overhang // Linear fragments with 1 cut that are no directional will always give a // 2 fragments - if len(overhangs) == 1 && !directional && !seq.Circular { // Check the case of a single cut + if len(overhangs) == 1 && !directional && !part.Circular { // Check the case of a single cut // In the case of a single cut in a linear sequence, we get two fragments with only 1 stick end - fragmentSeq1 := sequence[overhangs[0].Position+overhangs[0].Length:] - fragmentSeq2 := sequence[:overhangs[0].Position] - overhangSeq := sequence[overhangs[0].Position : overhangs[0].Position+overhangs[0].Length] - fragments = append(fragments, Fragment{fragmentSeq1, overhangSeq, ""}) - fragments = append(fragments, Fragment{fragmentSeq2, "", overhangSeq}) + fragmentSequence1 := sequence[overhangs[0].Position+overhangs[0].Length:] + fragmentSequence2 := sequence[:overhangs[0].Position] + overhangSequence := sequence[overhangs[0].Position : overhangs[0].Position+overhangs[0].Length] + fragments = append(fragments, Fragment{fragmentSequence1, overhangSequence, ""}) + fragments = append(fragments, Fragment{fragmentSequence2, "", overhangSequence}) return fragments } // Circular fragments with 1 cut will always have 2 overhangs (because of the // concat earlier). If we don't require directionality, this will always get // cut into a single fragment - if len(overhangs) == 2 && !directional && seq.Circular { + if len(overhangs) == 2 && !directional && part.Circular { // In the case of a single cut in a circular sequence, we get one fragment out with sticky overhangs - fragmentSeq1 := sequence[overhangs[0].Position+overhangs[0].Length : len(seq.Sequence)] - fragmentSeq2 := sequence[:overhangs[0].Position] - fragmentSeq := fragmentSeq1 + fragmentSeq2 - overhangSeq := sequence[overhangs[0].Position : overhangs[0].Position+overhangs[0].Length] - fragments = append(fragments, Fragment{fragmentSeq, overhangSeq, overhangSeq}) + fragmentSequence1 := sequence[overhangs[0].Position+overhangs[0].Length : len(part.Sequence)] + fragmentSequence2 := sequence[:overhangs[0].Position] + fragmentSequence := fragmentSequence1 + fragmentSequence2 + overhangSequence := sequence[overhangs[0].Position : overhangs[0].Position+overhangs[0].Length] + fragments = append(fragments, Fragment{fragmentSequence, overhangSequence, overhangSequence}) return fragments } @@ -205,28 +223,28 @@ func CutWithEnzyme(seq Part, directional bool, enzyme Enzyme) []Fragment { // the basis of GoldenGate assembly. if directional && !palindromic { if currentOverhang.Forward && !nextOverhang.Forward { - fragmentSeqs = append(fragmentSeqs, sequence[currentOverhang.Position:nextOverhang.Position]) + fragmentSequences = append(fragmentSequences, sequence[currentOverhang.Position:nextOverhang.Position]) } // We have to subtract RecognitionSitePlusSkipLength in case we have a recognition site on // one side of the origin of a circular sequence and the cut site on the other side of the origin - if nextOverhang.Position-nextOverhang.RecognitionSitePlusSkipLength > len(seq.Sequence) { + if nextOverhang.Position-nextOverhang.RecognitionSitePlusSkipLength > len(part.Sequence) { break } } else { - fragmentSeqs = append(fragmentSeqs, sequence[currentOverhang.Position:nextOverhang.Position]) - if nextOverhang.Position-nextOverhang.RecognitionSitePlusSkipLength > len(seq.Sequence) { + fragmentSequences = append(fragmentSequences, sequence[currentOverhang.Position:nextOverhang.Position]) + if nextOverhang.Position-nextOverhang.RecognitionSitePlusSkipLength > len(part.Sequence) { break } } } // Convert fragment sequences into fragments - for _, fragment := range fragmentSeqs { + for _, fragmentsequence := range fragmentSequences { // Minimum lengths (given oligos) for assembly is 8 base pairs // https://doi.org/10.1186/1756-0500-3-291 - if len(fragment) > 8 { - fragmentSequence := fragment[enzyme.OverhangLen : len(fragment)-enzyme.OverhangLen] - forwardOverhang := fragment[:enzyme.OverhangLen] - reverseOverhang := fragment[len(fragment)-enzyme.OverhangLen:] + if len(fragmentsequence) > 8 { + fragmentSequence := fragmentsequence[enzyme.OverheadLength : len(fragmentsequence)-enzyme.OverheadLength] + forwardOverhang := fragmentsequence[:enzyme.OverheadLength] + reverseOverhang := fragmentsequence[len(fragmentsequence)-enzyme.OverheadLength:] fragments = append(fragments, Fragment{Sequence: fragmentSequence, ForwardOverhang: forwardOverhang, ReverseOverhang: reverseOverhang}) } } @@ -235,94 +253,73 @@ func CutWithEnzyme(seq Part, directional bool, enzyme Enzyme) []Fragment { return fragments } -func recurseLigate(wg *sync.WaitGroup, constructs chan string, infiniteLoopingConstructs chan string, seedFragment Fragment, fragmentList []Fragment, usedFragments []Fragment) { +func recurseLigate(seedFragment Fragment, fragmentList []Fragment, usedFragments []Fragment, existingSeqhashes map[string]struct{}) (openConstructs []string, infiniteConstructs []string) { // Recurse ligate simulates all possible ligations of a series of fragments. Each possible combination begins with a "seed" that fragments from the pool can be added to. - defer wg.Done() // If the seed ligates to itself, we can call it done with a successful circularization! if seedFragment.ForwardOverhang == seedFragment.ReverseOverhang { - constructs <- seedFragment.ForwardOverhang + seedFragment.Sequence - } else { - for _, newFragment := range fragmentList { - // If the seedFragment's reverse overhang is ligates to a fragment's forward overhang, we can ligate those together and seed another ligation reaction - var newSeed Fragment - var fragmentAttached bool - if seedFragment.ReverseOverhang == newFragment.ForwardOverhang { - fragmentAttached = true - newSeed = Fragment{seedFragment.Sequence + seedFragment.ReverseOverhang + newFragment.Sequence, seedFragment.ForwardOverhang, newFragment.ReverseOverhang} - } - // This checks if we can ligate the next fragment in its reverse direction. We have to be careful though - if our seed has a palindrome, it will ligate to itself - // like [-> <- -> <- -> ...] infinitely. We check for that case here as well. - if (seedFragment.ReverseOverhang == transform.ReverseComplement(newFragment.ReverseOverhang)) && (seedFragment.ReverseOverhang != transform.ReverseComplement(seedFragment.ReverseOverhang)) { // If the second statement isn't there, program will crash on palindromes - fragmentAttached = true - newSeed = Fragment{seedFragment.Sequence + seedFragment.ReverseOverhang + transform.ReverseComplement(newFragment.Sequence), seedFragment.ForwardOverhang, transform.ReverseComplement(newFragment.ForwardOverhang)} - } - - // If fragment is actually attached, move to some checks - if fragmentAttached { - // If the newFragment's reverse complement already exists in the used fragment list, we need to cancel the recursion. - for _, usedFragment := range usedFragments { - if usedFragment.Sequence == newFragment.Sequence { - infiniteLoopingConstructs <- usedFragment.ForwardOverhang + usedFragment.Sequence + usedFragment.ReverseOverhang - return - } - } - wg.Add(1) - // If everything is clear, append fragment to usedFragments and recurse. - usedFragments = append(usedFragments, newFragment) - go recurseLigate(wg, constructs, infiniteLoopingConstructs, newSeed, fragmentList, usedFragments) - } + construct := seedFragment.ForwardOverhang + seedFragment.Sequence + seqhash, _ := seqhash.Hash(construct, "DNA", true, true) + if _, ok := existingSeqhashes[seqhash]; ok { + return nil, nil } + existingSeqhashes[seqhash] = struct{}{} + return []string{construct}, nil } -} -func getConstructs(c chan string, constructSequences chan []string, circular bool) { - var constructs []string - var exists bool - var existingSeqhashes []string - for { - construct, more := <-c - if more { - exists = false - seqhashConstruct, _ := seqhash.Hash(construct, "DNA", circular, true) - // Check if this construct is unique - for _, existingSeqhash := range existingSeqhashes { - if existingSeqhash == seqhashConstruct { - exists = true + // If the seed ligates to another fragment, we can recurse and add that fragment to the seed + for _, newFragment := range fragmentList { + // If the seedFragment's reverse overhang is ligates to a fragment's forward overhang, we can ligate those together and seed another ligation reaction + var newSeed Fragment + var fragmentAttached bool + if seedFragment.ReverseOverhang == newFragment.ForwardOverhang { + fragmentAttached = true + newSeed = Fragment{seedFragment.Sequence + seedFragment.ReverseOverhang + newFragment.Sequence, seedFragment.ForwardOverhang, newFragment.ReverseOverhang} + } + // This checks if we can ligate the next fragment in its reverse direction. We have to be careful though - if our seed has a palindrome, it will ligate to itself + // like [-> <- -> <- -> ...] infinitely. We check for that case here as well. + if (seedFragment.ReverseOverhang == transform.ReverseComplement(newFragment.ReverseOverhang)) && (seedFragment.ReverseOverhang != transform.ReverseComplement(seedFragment.ReverseOverhang)) { // If the second statement isn't there, program will crash on palindromes + fragmentAttached = true + newSeed = Fragment{seedFragment.Sequence + seedFragment.ReverseOverhang + transform.ReverseComplement(newFragment.Sequence), seedFragment.ForwardOverhang, transform.ReverseComplement(newFragment.ForwardOverhang)} + } + + // If fragment is actually attached, move to some checks + if fragmentAttached { + // If the newFragment's reverse complement already exists in the used fragment list, we need to cancel the recursion. + for _, usedFragment := range usedFragments { + if usedFragment.Sequence == newFragment.Sequence { + infiniteConstruct := usedFragment.ForwardOverhang + usedFragment.Sequence + usedFragment.ReverseOverhang + seqhash, _ := seqhash.Hash(infiniteConstruct, "DNA", false, true) + if _, ok := existingSeqhashes[seqhash]; ok { + return nil, nil + } + existingSeqhashes[seqhash] = struct{}{} + return nil, []string{infiniteConstruct} } } - if !exists { - constructs = append(constructs, construct) - existingSeqhashes = append(existingSeqhashes, seqhashConstruct) - } - } else { - constructSequences <- constructs - close(constructSequences) - return + // If everything is clear, append fragment to usedFragments and recurse. + usedFragments = append(usedFragments, newFragment) + openconstructs, infiniteconstructs := recurseLigate(newSeed, fragmentList, usedFragments, existingSeqhashes) + + openConstructs = append(openConstructs, openconstructs...) + infiniteConstructs = append(infiniteConstructs, infiniteconstructs...) } } + + return openConstructs, infiniteConstructs } // CircularLigate simulates ligation of all possible fragment combinations into circular plasmids. -func CircularLigate(fragments []Fragment) ([]string, []string, error) { - var wg sync.WaitGroup +func CircularLigate(fragments []Fragment) ([]string, []string) { var outputConstructs []string var outputInfiniteLoopingConstructs []string - constructs := make(chan string) - infiniteLoopingConstructs := make(chan string) // sometimes we will get stuck in infinite loops. These are sequences with a recursion break - constructSequences := make(chan []string) - infiniteLoopingConstructSequences := make(chan []string) + existingSeqhashes := make(map[string]struct{}) for _, fragment := range fragments { - wg.Add(1) - go recurseLigate(&wg, constructs, infiniteLoopingConstructs, fragment, fragments, []Fragment{}) + openConstructs, infiniteConstructs := recurseLigate(fragment, fragments, []Fragment{}, existingSeqhashes) + + outputConstructs = append(outputConstructs, openConstructs...) + outputInfiniteLoopingConstructs = append(outputInfiniteLoopingConstructs, infiniteConstructs...) } - go getConstructs(constructs, constructSequences, true) - go getConstructs(infiniteLoopingConstructs, infiniteLoopingConstructSequences, false) - wg.Wait() - close(constructs) - close(infiniteLoopingConstructs) - outputConstructs = <-constructSequences - outputInfiniteLoopingConstructs = <-infiniteLoopingConstructSequences - return outputConstructs, outputInfiniteLoopingConstructs, nil + return outputConstructs, outputInfiniteLoopingConstructs } /****************************************************************************** @@ -333,14 +330,21 @@ Specific cloning functions begin here. // GoldenGate simulates a GoldenGate cloning reaction. As of right now, we only // support BsaI, BbsI, BtgZI, and BsmBI. -func GoldenGate(sequences []Part, enzymeStr string) ([]string, []string, error) { +func GoldenGate(sequences []Part, cuttingEnzyme Enzyme) (openConstructs []string, infiniteLoops []string) { var fragments []Fragment for _, sequence := range sequences { - newFragments, err := CutWithEnzymeByName(sequence, true, enzymeStr) - if err != nil { - return []string{}, []string{}, err - } + newFragments := CutWithEnzyme(sequence, true, cuttingEnzyme) fragments = append(fragments, newFragments...) } - return CircularLigate(fragments) + openconstructs, infiniteloops := CircularLigate(fragments) + return openconstructs, infiniteloops +} + +// GetBaseRestrictionEnzymes return a basic slice of common enzymes used in Golden Gate Assembly. Eventually, we want to get the data for this map from ftp://ftp.neb.com/pub/rebase +func GetBaseRestrictionEnzymes() []Enzyme { + return []Enzyme{ + {"BsaI", regexp.MustCompile("GGTCTC"), regexp.MustCompile("GAGACC"), 1, 4, "GGTCTC"}, + {"BbsI", regexp.MustCompile("GAAGAC"), regexp.MustCompile("GTCTTC"), 2, 4, "GAAGAC"}, + {"BtgZI", regexp.MustCompile("GCGATG"), regexp.MustCompile("CATCGC"), 10, 4, "GCGATG"}, + } } diff --git a/clone/clone_test.go b/clone/clone_test.go index 2cfb57615..fb5ade198 100644 --- a/clone/clone_test.go +++ b/clone/clone_test.go @@ -1,55 +1,53 @@ -package clone_test +package clone import ( - "fmt" "testing" - - "github.com/TimothyStiles/poly/clone" - "github.com/TimothyStiles/poly/seqhash" ) // pOpen plasmid series (https://stanford.freegenes.org/collections/open-genes/products/open-plasmids#description). I use it for essentially all my cloning. -Keoni -var popen = clone.Part{"TAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGGCCTACTATTAGCAACAACGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACCTGCACCAGTCAGTAAAACGACGGCCAGTAGTCAAAAGCCTCCGACCGGAGGCTTTTGACTTGGTTCAGGTGGAGTGGGAGTAgtcttcGCcatcgCtACTAAAagccagataacagtatgcgtatttgcgcgctgatttttgcggtataagaatatatactgatatgtatacccgaagtatgtcaaaaagaggtatgctatgaagcagcgtattacagtgacagttgacagcgacagctatcagttgctcaaggcatatatgatgtcaatatctccggtctggtaagcacaaccatgcagaatgaagcccgtcgtctgcgtgccgaacgctggaaagcggaaaatcaggaagggatggctgaggtcgcccggtttattgaaatgaacggctcttttgctgacgagaacagggGCTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCCTTATACACAGgcgatgttgaagaccaCGCTGAGGTGTCAATCGTCGGAGCCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCATGGTCATAGCTGTTTCCTGAGAGCTTGGCAGGTGATGACACACATTAACAAATTTCGTGAGGAGTCTCCAGAAGAATGCCATTAATTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGG", true} +var popen = Part{"TAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGGCCTACTATTAGCAACAACGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACCTGCACCAGTCAGTAAAACGACGGCCAGTAGTCAAAAGCCTCCGACCGGAGGCTTTTGACTTGGTTCAGGTGGAGTGGGAGTAgtcttcGCcatcgCtACTAAAagccagataacagtatgcgtatttgcgcgctgatttttgcggtataagaatatatactgatatgtatacccgaagtatgtcaaaaagaggtatgctatgaagcagcgtattacagtgacagttgacagcgacagctatcagttgctcaaggcatatatgatgtcaatatctccggtctggtaagcacaaccatgcagaatgaagcccgtcgtctgcgtgccgaacgctggaaagcggaaaatcaggaagggatggctgaggtcgcccggtttattgaaatgaacggctcttttgctgacgagaacagggGCTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCCTTATACACAGgcgatgttgaagaccaCGCTGAGGTGTCAATCGTCGGAGCCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCATGGTCATAGCTGTTTCCTGAGAGCTTGGCAGGTGATGACACACATTAACAAATTTCGTGAGGAGTCTCCAGAAGAATGCCATTAATTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGG", true} func TestCutWithEnzymeByName(t *testing.T) { - _, err := clone.CutWithEnzymeByName(popen, true, "EcoFake") + enzymeManager := NewEnzymeManager(GetBaseRestrictionEnzymes()) + _, err := enzymeManager.CutWithEnzymeByName(popen, true, "EcoFake") if err == nil { t.Errorf("CutWithEnzymeByName should have failed when looking for fake restriction enzyme EcoFake") } } func TestCutWithEnzyme(t *testing.T) { - var seq clone.Part + enzymeManager := NewEnzymeManager(GetBaseRestrictionEnzymes()) + var sequence Part bsai := "GGTCTCAATGC" bsaiComplement := "ATGCAGAGACC" // test(1) // Test case of `<-bsaiComplement bsai-> <-bsaiComplement bsai->` where bsaI cuts off of a linear sequence. This tests the line: - // if !seq.Circular && (overhangSet[len(overhangSet)-1].Position+enzyme.EnzymeSkip+enzyme.EnzymeOverhangLen > len(sequence)) - seq = clone.Part{"ATATATA" + bsaiComplement + bsai + "ATGCATCGATCGACTAGCATG" + bsaiComplement + bsai[:8], false} - frag, err := clone.CutWithEnzymeByName(seq, true, "BsaI") + // if !sequence.Circular && (overhangSet[len(overhangSet)-1].Position+enzyme.EnzymeSkip+enzyme.EnzymeOverhangLen > len(sequence)) + sequence = Part{"ATATATA" + bsaiComplement + bsai + "ATGCATCGATCGACTAGCATG" + bsaiComplement + bsai[:8], false} + fragment, err := enzymeManager.CutWithEnzymeByName(sequence, true, "BsaI") if err != nil { t.Errorf("CutWithEnzyme should not have failed on test(1). Got error: %s", err) } - if len(frag) != 1 { + if len(fragment) != 1 { t.Errorf("CutWithEnzyme in test(1) should be 1 fragment in length") } - if frag[0].Sequence != "ATGCATCGATCGACTAGCATG" { - t.Errorf("CutWithEnzyme in test(1) should give fragment with sequence ATGCATCGATCGACTAGCATG . Got sequence: %s", frag[0].Sequence) + if fragment[0].Sequence != "ATGCATCGATCGACTAGCATG" { + t.Errorf("CutWithEnzyme in test(1) should give fragment with sequence ATGCATCGATCGACTAGCATG . Got sequence: %s", fragment[0].Sequence) } // test(2) // Now if we take the same sequence and circularize it, we get a different result - seq.Circular = true - frag, err = clone.CutWithEnzymeByName(seq, true, "BsaI") + sequence.Circular = true + fragment, err = enzymeManager.CutWithEnzymeByName(sequence, true, "BsaI") if err != nil { t.Errorf("CutWithEnzyme should not have failed on test(2). Got error: %s", err) } - if len(frag) != 2 { + if len(fragment) != 2 { t.Errorf("CutWithEnzyme in test(2) should be 1 fragment in length") } - if frag[0].Sequence != "ATGCATCGATCGACTAGCATG" || frag[1].Sequence != "TATA" { - t.Errorf("CutWithEnzyme in test(2) should give fragment with sequence ATGCATCGATCGACTAGCATG and TATA. Got sequence: %s and %s", frag[0].Sequence, frag[1].Sequence) + if fragment[0].Sequence != "ATGCATCGATCGACTAGCATG" || fragment[1].Sequence != "TATA" { + t.Errorf("CutWithEnzyme in test(2) should give fragment with sequence ATGCATCGATCGACTAGCATG and TATA. Got sequence: %s and %s", fragment[0].Sequence, fragment[1].Sequence) } // test(3) @@ -57,43 +55,43 @@ func TestCutWithEnzyme(t *testing.T) { // different results if we have a linear or circular DNA. Since single cuts // will give no fragments if you test for directionality, we set the // directionality flag to false. This tests the line: - // if len(overhangs) == 1 && !directional && !seq.Circular - seq = clone.Part{"ATATATATATATATAT" + bsai + "GCGCGCGCGCGCGCGCGCGC", false} - frag, err = clone.CutWithEnzymeByName(seq, false, "BsaI") + // if len(overhangs) == 1 && !directional && !sequence.Circular + sequence = Part{"ATATATATATATATAT" + bsai + "GCGCGCGCGCGCGCGCGCGC", false} + fragment, err = enzymeManager.CutWithEnzymeByName(sequence, false, "BsaI") if err != nil { t.Errorf("CutWithEnzyme should not have failed on test(3). Got error: %s", err) } - if len(frag) != 2 { + if len(fragment) != 2 { t.Errorf("Cutting a linear fragment with a single cut site should give 2 fragments") } - if frag[0].Sequence != "GCGCGCGCGCGCGCGCGCGC" || frag[1].Sequence != "ATATATATATATATATGGTCTCA" { - t.Errorf("CutWithEnzyme in test(3) should give fragment with sequence GCGCGCGCGCGCGCGCGCGC and ATATATATATATATATGGTCTCA. Got sequence: %s and %s", frag[0].Sequence, frag[1].Sequence) + if fragment[0].Sequence != "GCGCGCGCGCGCGCGCGCGC" || fragment[1].Sequence != "ATATATATATATATATGGTCTCA" { + t.Errorf("CutWithEnzyme in test(3) should give fragment with sequence GCGCGCGCGCGCGCGCGCGC and ATATATATATATATATGGTCTCA. Got sequence: %s and %s", fragment[0].Sequence, fragment[1].Sequence) } // test(4) // This tests for the above except with a circular fragment. Specifically, it // tests the line: - // if len(overhangs) == 2 && !directional && seq.Circular - seq.Circular = true - frag, err = clone.CutWithEnzymeByName(seq, false, "BsaI") + // if len(overhangs) == 2 && !directional && sequence.Circular + sequence.Circular = true + fragment, err = enzymeManager.CutWithEnzymeByName(sequence, false, "BsaI") if err != nil { t.Errorf("CutWithEnzyme should not have failed on test(4). Got error: %s", err) } - if len(frag) != 1 { + if len(fragment) != 1 { t.Errorf("Cutting a circular fragment with a single cut site should give 1 fragments") } - if frag[0].Sequence != "GCGCGCGCGCGCGCGCGCGCATATATATATATATATGGTCTCA" { - t.Errorf("CutWithEnzyme in test(4) should give fragment with sequence ATATATATATATATATGGTCTCA. Got Sequence: %s", frag[0].Sequence) + if fragment[0].Sequence != "GCGCGCGCGCGCGCGCGCGCATATATATATATATATGGTCTCA" { + t.Errorf("CutWithEnzyme in test(4) should give fragment with sequence ATATATATATATATATGGTCTCA. Got Sequence: %s", fragment[0].Sequence) } // test(5) // This tests if we have a fragment where we do not care about directionality // but have more than 1 cut site in our fragment. We can use pOpen for this. - frag, err = clone.CutWithEnzymeByName(popen, false, "BbsI") + fragment, err = enzymeManager.CutWithEnzymeByName(popen, false, "BbsI") if err != nil { t.Errorf("CutWithEnzyme should not have failed on test(5). Got error: %s", err) } - if len(frag) != 2 { + if len(fragment) != 2 { t.Errorf("Cutting pOpen without a direction should yield 2 fragments") } } @@ -101,12 +99,9 @@ func TestCutWithEnzyme(t *testing.T) { func TestCircularLigate(t *testing.T) { // The following tests for complementing overhangs. Specific, this line: // newSeed := Fragment{seedFragment.Sequence + seedFragment.ReverseOverhang + ReverseComplement(newFragment.Sequence), seedFragment.ForwardOverhang, ReverseComplement(newFragment.ForwardOverhang)} - fragment1 := clone.Fragment{"AAAAAA", "GTTG", "CTAT"} - fragment2 := clone.Fragment{"AAAAAA", "CAAC", "ATAG"} - outputConstructs, infiniteLoops, err := clone.CircularLigate([]clone.Fragment{fragment1, fragment2}) - if err != nil { - t.Errorf("Failed circular ligation with error: %s", err) - } + fragment1 := Fragment{"AAAAAA", "GTTG", "CTAT"} + fragment2 := Fragment{"AAAAAA", "CAAC", "ATAG"} + outputConstructs, infiniteLoops := CircularLigate([]Fragment{fragment1, fragment2}) if len(outputConstructs) != 1 { t.Errorf("Circular ligation with complementing overhangs should only output 1 valid rotated sequence.") } @@ -115,53 +110,39 @@ func TestCircularLigate(t *testing.T) { } } -func TestGoldenGate(t *testing.T) { - // Here we test if the enzyme we want to use in a GoldenGate reaction does not exist in our enzyme pool - fragment1 := clone.Part{"GAAGTGCCATTCCGCCTGACCTGAAGACCAGGAGAAACACGTGGCAAACATTCCGGTCTCAAATGGAAAAGAGCAACGAAACCAACGGCTACCTTGACAGCGCTCAAGCCGGCCCTGCAGCTGGCCCGGGCGCTCCGGGTACCGCCGCGGGTCGTGCACGTCGTTGCGCGGGCTTCCTGCGGCGCCAAGCGCTGGTGCTGCTCACGGTGTCTGGTGTTCTGGCAGGCGCCGGTTTGGGCGCGGCACTGCGTGGGCTCAGCCTGAGCCGCACCCAGGTCACCTACCTGGCCTTCCCCGGCGAGATGCTGCTCCGCATGCTGCGCATGATCATCCTGCCGCTGGTGGTCTGCAGCCTGGTGTCGGGCGCCGCCTCCCTCGATGCCAGCTGCCTCGGGCGTCTGGGCGGTATCGCTGTCGCCTACTTTGGCCTCACCACACTGAGTGCCTCGGCGCTCGCCGTGGCCTTGGCGTTCATCATCAAGCCAGGATCCGGTGCGCAGACCCTTCAGTCCAGCGACCTGGGGCTGGAGGACTCGGGGCCTCCTCCTGTCCCCAAAGAAACGGTGGACTCTTTCCTCGACCTGGCCAGAAACCTGTTTCCCTCCAATCTTGTGGTTGCAGCTTTCCGTACGTATGCAACCGATTATAAAGTCGTGACCCAGAACAGCAGCTCTGGAAATGTAACCCATGAAAAGATCCCCATAGGCACTGAGATAGAAGGGATGAACATTTTAGGATTGGTCCTGTTTGCTCTGGTGTTAGGAGTGGCCTTAAAGAAACTAGGCTCCGAAGGAGAGGACCTCATCCGTTTCTTCAATTCCCTCAACGAGGCGACGATGGTGCTGGTGTCCTGGATTATGTGGTACGCGTCTTCAGGCTAGGTGGAGGCTCAGTG", false} - fragment2 := clone.Part{"GAAGTGCCATTCCGCCTGACCTGAAGACCAGTACGTACCTGTGGGCATCATGTTCCTTGTTGGAAGCAAGATCGTGGAAATGAAAGACATCATCGTGCTGGTGACCAGCCTGGGGAAATACATCTTCGCATCTATATTGGGCCACGTCATTCATGGTGGTATCGTCCTGCCGCTGATTTATTTTGTTTTCACACGAAAAAACCCATTCAGATTCCTCCTGGGCCTCCTCGCCCCATTTGCGACAGCATTTGCTACGTGCTCCAGCTCAGCGACCCTTCCCTCTATGATGAAGTGCATTGAAGAGAACAATGGTGTGGACAAGAGGATCTCCAGGTTTATTCTCCCCATCGGGGCCACCGTGAACATGGACGGAGCAGCCATCTTCCAGTGTGTGGCCGCGGTGTTCATTGCGCAACTCAACAACGTAGAGCTCAACGCAGGACAGATTTTCACCATTCTAGTGACTGCCACAGCGTCCAGTGTTGGAGCAGCAGGCGTGCCAGCTGGAGGGGTCCTCACCATTGCCATTATCCTGGAGGCCATTGGGCTGCCTACTCATGATCTGCCTCTGATCCTGGCTGTGGACTGGATTGTGGACCGGACCACCACGGTGGTGAATGTGGAAGGGGATGCCCTGGGTGCAGGCATTCTCCACCACCTGAATCAGAAGGCAACAAAGAAAGGCGAGCAGGAACTTGCTGAGGTGAAAGTGGAAGCCATCCCCAACTGCAAGTCTGAGGAGGAAACCTCGCCCCTGGTGACACACCAGAACCCCGCTGGCCCCGTGGCCAGTGCCCCAGAACTGGAATCCAAGGAGTCGGTTCTGTGAAGAGCTTAGAGACCGACGACTGCCTAAGGACATTCGCTGCGTCTTCAGGCTAGGTGGAGGCTCAGTG", false} - - _, _, err := clone.GoldenGate([]clone.Part{fragment1, fragment2, popen}, "EcoRFake") +func TestEnzymeManage_GetEnzymeByName_NotFound(t *testing.T) { + enzymeManager := NewEnzymeManager(GetBaseRestrictionEnzymes()) + _, err := enzymeManager.GetEnzymeByName("EcoRFake") if err == nil { t.Errorf("GoldenGate should fail when using enzyme EcoRFake") } - if err.Error() != "Enzyme EcoRFake not found in enzymeMap" { + if err.Error() != "Enzyme EcoRFake not found" { t.Errorf("Failure of GoldenGate on incorrect enzyme should follow the exact string `Enzyme EcoRFake not found in enzymeMap`. Got: %s", err.Error()) } } -func ExampleGoldenGate() { - // Fragment 1 has a palindrome at its start. This isn't very common but - // can occur. These two fragments are real DNA fragments used in the - // FreeGenes Project. They are used because they were on my computer - // - Keoni - fragment1 := clone.Part{"GAAGTGCCATTCCGCCTGACCTGAAGACCAGGAGAAACACGTGGCAAACATTCCGGTCTCAAATGGAAAAGAGCAACGAAACCAACGGCTACCTTGACAGCGCTCAAGCCGGCCCTGCAGCTGGCCCGGGCGCTCCGGGTACCGCCGCGGGTCGTGCACGTCGTTGCGCGGGCTTCCTGCGGCGCCAAGCGCTGGTGCTGCTCACGGTGTCTGGTGTTCTGGCAGGCGCCGGTTTGGGCGCGGCACTGCGTGGGCTCAGCCTGAGCCGCACCCAGGTCACCTACCTGGCCTTCCCCGGCGAGATGCTGCTCCGCATGCTGCGCATGATCATCCTGCCGCTGGTGGTCTGCAGCCTGGTGTCGGGCGCCGCCTCCCTCGATGCCAGCTGCCTCGGGCGTCTGGGCGGTATCGCTGTCGCCTACTTTGGCCTCACCACACTGAGTGCCTCGGCGCTCGCCGTGGCCTTGGCGTTCATCATCAAGCCAGGATCCGGTGCGCAGACCCTTCAGTCCAGCGACCTGGGGCTGGAGGACTCGGGGCCTCCTCCTGTCCCCAAAGAAACGGTGGACTCTTTCCTCGACCTGGCCAGAAACCTGTTTCCCTCCAATCTTGTGGTTGCAGCTTTCCGTACGTATGCAACCGATTATAAAGTCGTGACCCAGAACAGCAGCTCTGGAAATGTAACCCATGAAAAGATCCCCATAGGCACTGAGATAGAAGGGATGAACATTTTAGGATTGGTCCTGTTTGCTCTGGTGTTAGGAGTGGCCTTAAAGAAACTAGGCTCCGAAGGAGAGGACCTCATCCGTTTCTTCAATTCCCTCAACGAGGCGACGATGGTGCTGGTGTCCTGGATTATGTGGTACGCGTCTTCAGGCTAGGTGGAGGCTCAGTG", false} - fragment2 := clone.Part{"GAAGTGCCATTCCGCCTGACCTGAAGACCAGTACGTACCTGTGGGCATCATGTTCCTTGTTGGAAGCAAGATCGTGGAAATGAAAGACATCATCGTGCTGGTGACCAGCCTGGGGAAATACATCTTCGCATCTATATTGGGCCACGTCATTCATGGTGGTATCGTCCTGCCGCTGATTTATTTTGTTTTCACACGAAAAAACCCATTCAGATTCCTCCTGGGCCTCCTCGCCCCATTTGCGACAGCATTTGCTACGTGCTCCAGCTCAGCGACCCTTCCCTCTATGATGAAGTGCATTGAAGAGAACAATGGTGTGGACAAGAGGATCTCCAGGTTTATTCTCCCCATCGGGGCCACCGTGAACATGGACGGAGCAGCCATCTTCCAGTGTGTGGCCGCGGTGTTCATTGCGCAACTCAACAACGTAGAGCTCAACGCAGGACAGATTTTCACCATTCTAGTGACTGCCACAGCGTCCAGTGTTGGAGCAGCAGGCGTGCCAGCTGGAGGGGTCCTCACCATTGCCATTATCCTGGAGGCCATTGGGCTGCCTACTCATGATCTGCCTCTGATCCTGGCTGTGGACTGGATTGTGGACCGGACCACCACGGTGGTGAATGTGGAAGGGGATGCCCTGGGTGCAGGCATTCTCCACCACCTGAATCAGAAGGCAACAAAGAAAGGCGAGCAGGAACTTGCTGAGGTGAAAGTGGAAGCCATCCCCAACTGCAAGTCTGAGGAGGAAACCTCGCCCCTGGTGACACACCAGAACCCCGCTGGCCCCGTGGCCAGTGCCCCAGAACTGGAATCCAAGGAGTCGGTTCTGTGAAGAGCTTAGAGACCGACGACTGCCTAAGGACATTCGCTGCGTCTTCAGGCTAGGTGGAGGCTCAGTG", false} - - Clones, _, _ := clone.GoldenGate([]clone.Part{fragment1, fragment2, popen}, "BbsI") - - fmt.Println(seqhash.RotateSequence(Clones[0])) - // Output: AAAAAAAGGATCTCAAGAAGGCCTACTATTAGCAACAACGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACCTGCACCAGTCAGTAAAACGACGGCCAGTAGTCAAAAGCCTCCGACCGGAGGCTTTTGACTTGGTTCAGGTGGAGTGGGAGAAACACGTGGCAAACATTCCGGTCTCAAATGGAAAAGAGCAACGAAACCAACGGCTACCTTGACAGCGCTCAAGCCGGCCCTGCAGCTGGCCCGGGCGCTCCGGGTACCGCCGCGGGTCGTGCACGTCGTTGCGCGGGCTTCCTGCGGCGCCAAGCGCTGGTGCTGCTCACGGTGTCTGGTGTTCTGGCAGGCGCCGGTTTGGGCGCGGCACTGCGTGGGCTCAGCCTGAGCCGCACCCAGGTCACCTACCTGGCCTTCCCCGGCGAGATGCTGCTCCGCATGCTGCGCATGATCATCCTGCCGCTGGTGGTCTGCAGCCTGGTGTCGGGCGCCGCCTCCCTCGATGCCAGCTGCCTCGGGCGTCTGGGCGGTATCGCTGTCGCCTACTTTGGCCTCACCACACTGAGTGCCTCGGCGCTCGCCGTGGCCTTGGCGTTCATCATCAAGCCAGGATCCGGTGCGCAGACCCTTCAGTCCAGCGACCTGGGGCTGGAGGACTCGGGGCCTCCTCCTGTCCCCAAAGAAACGGTGGACTCTTTCCTCGACCTGGCCAGAAACCTGTTTCCCTCCAATCTTGTGGTTGCAGCTTTCCGTACGTATGCAACCGATTATAAAGTCGTGACCCAGAACAGCAGCTCTGGAAATGTAACCCATGAAAAGATCCCCATAGGCACTGAGATAGAAGGGATGAACATTTTAGGATTGGTCCTGTTTGCTCTGGTGTTAGGAGTGGCCTTAAAGAAACTAGGCTCCGAAGGAGAGGACCTCATCCGTTTCTTCAATTCCCTCAACGAGGCGACGATGGTGCTGGTGTCCTGGATTATGTGGTACGTACCTGTGGGCATCATGTTCCTTGTTGGAAGCAAGATCGTGGAAATGAAAGACATCATCGTGCTGGTGACCAGCCTGGGGAAATACATCTTCGCATCTATATTGGGCCACGTCATTCATGGTGGTATCGTCCTGCCGCTGATTTATTTTGTTTTCACACGAAAAAACCCATTCAGATTCCTCCTGGGCCTCCTCGCCCCATTTGCGACAGCATTTGCTACGTGCTCCAGCTCAGCGACCCTTCCCTCTATGATGAAGTGCATTGAAGAGAACAATGGTGTGGACAAGAGGATCTCCAGGTTTATTCTCCCCATCGGGGCCACCGTGAACATGGACGGAGCAGCCATCTTCCAGTGTGTGGCCGCGGTGTTCATTGCGCAACTCAACAACGTAGAGCTCAACGCAGGACAGATTTTCACCATTCTAGTGACTGCCACAGCGTCCAGTGTTGGAGCAGCAGGCGTGCCAGCTGGAGGGGTCCTCACCATTGCCATTATCCTGGAGGCCATTGGGCTGCCTACTCATGATCTGCCTCTGATCCTGGCTGTGGACTGGATTGTGGACCGGACCACCACGGTGGTGAATGTGGAAGGGGATGCCCTGGGTGCAGGCATTCTCCACCACCTGAATCAGAAGGCAACAAAGAAAGGCGAGCAGGAACTTGCTGAGGTGAAAGTGGAAGCCATCCCCAACTGCAAGTCTGAGGAGGAAACCTCGCCCCTGGTGACACACCAGAACCCCGCTGGCCCCGTGGCCAGTGCCCCAGAACTGGAATCCAAGGAGTCGGTTCTGTGAAGAGCTTAGAGACCGACGACTGCCTAAGGACATTCGCTGAGGTGTCAATCGTCGGAGCCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCATGGTCATAGCTGTTTCCTGAGAGCTTGGCAGGTGATGACACACATTAACAAATTTCGTGAGGAGTCTCCAGAAGAATGCCATTAATTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAG -} - func TestSignalKilledGoldenGate(t *testing.T) { + enzymeManager := NewEnzymeManager(GetBaseRestrictionEnzymes()) // This previously would crash from using too much RAM. - frag1 := clone.Part{"AAAGCACTCTTAGGCCTCTGGAAGACATGGAGGGTCTCAAGGTGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTTTTGCCCTGTAAACGAAAAAACCACCTGGGTAGTCTTCGCATTTCTTAATCGGTGCCC", false} - frag2 := clone.Part{"AAAGCACTCTTAGGCCTCTGGAAGACATTGGGGAGGTGGTTTGATCGAAGGTTAAGTCAGTTGGGGAACTGCTTAACCGTGGTAACTGGCTTTCGCAGAGCACAGCAACCAAATCTGTTAGTCTTCGCATTTCTTAATCGGTGCCC", false} - frag3 := clone.Part{"AAAGCACTCTTAGGCCTCTGGAAGACATCTGTCCTTCCAGTGTAGCCGGACTTTGGCGCACACTTCAAGAGCAACCGCGTGTTTAGCTAAACAAATCCTCTGCGAACTCCCAGTTACCTAGTCTTCGCATTTCTTAATCGGTGCCC", false} - frag4 := clone.Part{"AAAGCACTCTTAGGCCTCTGGAAGACATTACCAATGGCTGCTGCCAGTGGCGTTTTACCGTGCTTTTCCGGGTTGGACTCAAGTGAACAGTTACCGGATAAGGCGCAGCAGTCGGGCTTAGTCTTCGCATTTCTTAATCGGTGCCC", false} - frag5 := clone.Part{"AAAGCACTCTTAGGCCTCTGGAAGACATGGCTGAACGGGGAGTTCTTGCTTACAGCCCAGCTTGGAGCGAACGACCTACACCGAGCCGAGATACCAGTGTGTGAGCTATGAGAAAGCGTAGTCTTCGCATTTCTTAATCGGTGCCC", false} - frag6 := clone.Part{"AAAGCACTCTTAGGCCTCTGGAAGACATAGCGCCACACTTCCCGTAAGGGAGAAAGGCGGAACAGGTATCCGGTAAACGGCAGGGTCGGAACAGGAGAGCGCAAGAGGGAGCGACCCGTAGTCTTCGCATTTCTTAATCGGTGCCC", false} - frag7 := clone.Part{"AAAGCACTCTTAGGCCTCTGGAAGACATCCCGCCGGAAACGGTGGGGATCTTTAAGTCCTGTCGGGTTTCGCCCGTACTGTCAGATTCATGGTTGAGCCTCACGGCTCCCACAGATGTAGTCTTCGCATTTCTTAATCGGTGCCC", false} - frag8 := clone.Part{"AAAGCACTCTTAGGCCTCTGGAAGACATGATGCACCGGAAAAGCGTCTGTTTATGTGAACTCTGGCAGGAGGGCGGAGCCTATGGAAAAACGCCACCGGCGCGGCCCTGCTGTTTTGCCTCACATGTTAGTCTTCGCATTTCTTAATCGGTGCCC", false} - frag9 := clone.Part{"AAAGCACTCTTAGGCCTCTGGAAGACATATGTTAGTCCCCTGCTTATCCACGGAATCTGTGGGTAACTTTGTATGTGTCCGCAGCGCAAAAAGAGACCCGCTTAGTCTTCGCATTTCTTAATCGGTGCCC", false} - fragments := []clone.Part{popen, frag1, frag2, frag3, frag4, frag5, frag6, frag7, frag8, frag9} - - clones, loopingClones, err := clone.GoldenGate(fragments, "BbsI") + fragment1 := Part{"AAAGCACTCTTAGGCCTCTGGAAGACATGGAGGGTCTCAAGGTGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTTTTGCCCTGTAAACGAAAAAACCACCTGGGTAGTCTTCGCATTTCTTAATCGGTGCCC", false} + fragment2 := Part{"AAAGCACTCTTAGGCCTCTGGAAGACATTGGGGAGGTGGTTTGATCGAAGGTTAAGTCAGTTGGGGAACTGCTTAACCGTGGTAACTGGCTTTCGCAGAGCACAGCAACCAAATCTGTTAGTCTTCGCATTTCTTAATCGGTGCCC", false} + fragment3 := Part{"AAAGCACTCTTAGGCCTCTGGAAGACATCTGTCCTTCCAGTGTAGCCGGACTTTGGCGCACACTTCAAGAGCAACCGCGTGTTTAGCTAAACAAATCCTCTGCGAACTCCCAGTTACCTAGTCTTCGCATTTCTTAATCGGTGCCC", false} + fragment4 := Part{"AAAGCACTCTTAGGCCTCTGGAAGACATTACCAATGGCTGCTGCCAGTGGCGTTTTACCGTGCTTTTCCGGGTTGGACTCAAGTGAACAGTTACCGGATAAGGCGCAGCAGTCGGGCTTAGTCTTCGCATTTCTTAATCGGTGCCC", false} + fragment5 := Part{"AAAGCACTCTTAGGCCTCTGGAAGACATGGCTGAACGGGGAGTTCTTGCTTACAGCCCAGCTTGGAGCGAACGACCTACACCGAGCCGAGATACCAGTGTGTGAGCTATGAGAAAGCGTAGTCTTCGCATTTCTTAATCGGTGCCC", false} + fragment6 := Part{"AAAGCACTCTTAGGCCTCTGGAAGACATAGCGCCACACTTCCCGTAAGGGAGAAAGGCGGAACAGGTATCCGGTAAACGGCAGGGTCGGAACAGGAGAGCGCAAGAGGGAGCGACCCGTAGTCTTCGCATTTCTTAATCGGTGCCC", false} + fragment7 := Part{"AAAGCACTCTTAGGCCTCTGGAAGACATCCCGCCGGAAACGGTGGGGATCTTTAAGTCCTGTCGGGTTTCGCCCGTACTGTCAGATTCATGGTTGAGCCTCACGGCTCCCACAGATGTAGTCTTCGCATTTCTTAATCGGTGCCC", false} + fragment8 := Part{"AAAGCACTCTTAGGCCTCTGGAAGACATGATGCACCGGAAAAGCGTCTGTTTATGTGAACTCTGGCAGGAGGGCGGAGCCTATGGAAAAACGCCACCGGCGCGGCCCTGCTGTTTTGCCTCACATGTTAGTCTTCGCATTTCTTAATCGGTGCCC", false} + fragment9 := Part{"AAAGCACTCTTAGGCCTCTGGAAGACATATGTTAGTCCCCTGCTTATCCACGGAATCTGTGGGTAACTTTGTATGTGTCCGCAGCGCAAAAAGAGACCCGCTTAGTCTTCGCATTTCTTAATCGGTGCCC", false} + fragments := []Part{popen, fragment1, fragment2, fragment3, fragment4, fragment5, fragment6, fragment7, fragment8, fragment9} + + bbsI, err := enzymeManager.GetEnzymeByName("BbsI") if err != nil { - t.Errorf("GoldenGate should not fail with these fragments. Got error: %s", err) + t.Errorf("Error when getting Enzyme. Got error: %s", err) } + + clones, loopingClones := GoldenGate(fragments, bbsI) if len(clones) != 1 { - t.Errorf("There should be 1 output clone. Got: %d", len(clones)) + t.Errorf("There should be 1 output Got: %d", len(clones)) } // This should be changed later when we have a better way of informing user of reused overhangs if len(loopingClones) != 4 { @@ -170,27 +151,31 @@ func TestSignalKilledGoldenGate(t *testing.T) { } func TestPanicGoldenGate(t *testing.T) { + enzymeManager := NewEnzymeManager(GetBaseRestrictionEnzymes()) // This used to panic with the message: // panic: runtime error: slice bounds out of range [:-2] [recovered] // It was from the following sequence: GAAGACATAATGGTCTTC . There are 2 intercepting BbsI sites. - frag1 := clone.Part{"AAACCGGAGCCATACAGTACGAAGACATGGAGGGTCTCAAATGAAAAAAATCATCGAAACCCAGCGTGCACCGGGAGCAATCGGACCGTACGTCCAGGGAGTCGACCTAGGATCAATGTAGTCTTCGCACTTGGCTTAGATGCAAC", false} - frag2 := clone.Part{"AAACCGGAGCCATACAGTACGAAGACATAATGGTCTTCACCTCAGGACAGATCCCGGTCTGCCCGCAGACCGGAGAAATCCCGGCAGACGTCCAGGACCAGGCACGTCTATCACTAGATAGTCTTCGCACTTGGCTTAGATGCAAC", false} - frag3 := clone.Part{"AAACCGGAGCCATACAGTACGAAGACATTAGAAAACGTCAAAGCAATCGTCGTCGCAGCAGGACTATCAGTCGGAGACATCATCAAAATGACCGTCTTCATCACCGACCTAAACGACTTAGTCTTCGCACTTGGCTTAGATGCAAC", false} - frag4 := clone.Part{"AAACCGGAGCCATACAGTACGAAGACATGACTTCGCAACCATCAACGAAGTCTACAAACAGTTCTTCGACGAACACCAGGCAACCTACCCGACCCGTTCATGCGTCCAGGTCGCACGTCTACTAGTCTTCGCACTTGGCTTAGATGCAAC", false} - frag5 := clone.Part{"AAACCGGAGCCATACAGTACGAAGACATCTACCGAAAGACGTCAAACTAGAAATCGAAGCAATCGCAGTCCGTTCAGCAAGAGCTTAGAGACCCGCTTAGTCTTCGCACTTGGCTTAGATGCAAC", false} - fragments := []clone.Part{popen, frag1, frag2, frag3, frag4, frag5} - - _, _, err := clone.GoldenGate(fragments, "BbsI") + fragment1 := Part{"AAACCGGAGCCATACAGTACGAAGACATGGAGGGTCTCAAATGAAAAAAATCATCGAAACCCAGCGTGCACCGGGAGCAATCGGACCGTACGTCCAGGGAGTCGACCTAGGATCAATGTAGTCTTCGCACTTGGCTTAGATGCAAC", false} + fragment2 := Part{"AAACCGGAGCCATACAGTACGAAGACATAATGGTCTTCACCTCAGGACAGATCCCGGTCTGCCCGCAGACCGGAGAAATCCCGGCAGACGTCCAGGACCAGGCACGTCTATCACTAGATAGTCTTCGCACTTGGCTTAGATGCAAC", false} + fragment3 := Part{"AAACCGGAGCCATACAGTACGAAGACATTAGAAAACGTCAAAGCAATCGTCGTCGCAGCAGGACTATCAGTCGGAGACATCATCAAAATGACCGTCTTCATCACCGACCTAAACGACTTAGTCTTCGCACTTGGCTTAGATGCAAC", false} + fragment4 := Part{"AAACCGGAGCCATACAGTACGAAGACATGACTTCGCAACCATCAACGAAGTCTACAAACAGTTCTTCGACGAACACCAGGCAACCTACCCGACCCGTTCATGCGTCCAGGTCGCACGTCTACTAGTCTTCGCACTTGGCTTAGATGCAAC", false} + fragment5 := Part{"AAACCGGAGCCATACAGTACGAAGACATCTACCGAAAGACGTCAAACTAGAAATCGAAGCAATCGCAGTCCGTTCAGCAAGAGCTTAGAGACCCGCTTAGTCTTCGCACTTGGCTTAGATGCAAC", false} + fragments := []Part{popen, fragment1, fragment2, fragment3, fragment4, fragment5} + + bbsI, err := enzymeManager.GetEnzymeByName("BbsI") if err != nil { - t.Errorf("GoldenGate should not fail with these fragments. Got error: %s", err) + t.Errorf("Error when getting Enzyme. Got error: %s", err) } + + _, _ = GoldenGate(fragments, bbsI) } func TestCircularCutRegression(t *testing.T) { + enzymeManager := NewEnzymeManager(GetBaseRestrictionEnzymes()) // This used to error with 0 fragments since the BsaI cut site is on the other // side of the origin from its recognition site. - plasmid1 := clone.Part{"AAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCCGAGaccaagtcgcggccgcgaggtgtcaatcgtcggagtagggataacagggtaatccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgcatggtcatagctgtttcctgttacgccccgccctgccactcgtcgcagtactgttgtaattcattaagcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgccagcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattggctgacacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagggatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccatccttcattgccatacgaaattccggatgagcattcatcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatccagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgccgatcatttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagtaaaacgacggccagtagtcaaaagcctccgaccggaggcttttgacttggttcaggtggagtggcggccgcgacttgGTCTC", true} - newFragments, err := clone.CutWithEnzymeByName(plasmid1, true, "BsaI") + plasmid1 := Part{"AAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCCGAGaccaagtcgcggccgcgaggtgtcaatcgtcggagtagggataacagggtaatccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgcatggtcatagctgtttcctgttacgccccgccctgccactcgtcgcagtactgttgtaattcattaagcattctgccgacatggaagccatcacaaacggcatgatgaacctgaatcgccagcggcatcagcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattggctgacacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagggatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccatccttcattgccatacgaaattccggatgagcattcatcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatccagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgccgatcatttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagtaaaacgacggccagtagtcaaaagcctccgaccggaggcttttgacttggttcaggtggagtggcggccgcgacttgGTCTC", true} + newFragments, err := enzymeManager.CutWithEnzymeByName(plasmid1, true, "BsaI") if err != nil { t.Errorf("Failed to cut: %s", err) } @@ -198,3 +183,21 @@ func TestCircularCutRegression(t *testing.T) { t.Errorf("Expected 1 new fragment, got: %d", len(newFragments)) } } + +func benchmarkGoldenGate(b *testing.B, enzymeManager EnzymeManager, parts []Part) { + bbsI, err := enzymeManager.GetEnzymeByName("BbsI") + if err != nil { + b.Errorf("Error when getting Enzyme. Got error: %s", err) + } + for n := 0; n < b.N; n++ { + _, _ = GoldenGate(parts, bbsI) + } +} + +func BenchmarkGoldenGate3Parts(b *testing.B) { + enzymeManager := NewEnzymeManager(GetBaseRestrictionEnzymes()) + fragment1 := Part{"GAAGTGCCATTCCGCCTGACCTGAAGACCAGGAGAAACACGTGGCAAACATTCCGGTCTCAAATGGAAAAGAGCAACGAAACCAACGGCTACCTTGACAGCGCTCAAGCCGGCCCTGCAGCTGGCCCGGGCGCTCCGGGTACCGCCGCGGGTCGTGCACGTCGTTGCGCGGGCTTCCTGCGGCGCCAAGCGCTGGTGCTGCTCACGGTGTCTGGTGTTCTGGCAGGCGCCGGTTTGGGCGCGGCACTGCGTGGGCTCAGCCTGAGCCGCACCCAGGTCACCTACCTGGCCTTCCCCGGCGAGATGCTGCTCCGCATGCTGCGCATGATCATCCTGCCGCTGGTGGTCTGCAGCCTGGTGTCGGGCGCCGCCTCCCTCGATGCCAGCTGCCTCGGGCGTCTGGGCGGTATCGCTGTCGCCTACTTTGGCCTCACCACACTGAGTGCCTCGGCGCTCGCCGTGGCCTTGGCGTTCATCATCAAGCCAGGATCCGGTGCGCAGACCCTTCAGTCCAGCGACCTGGGGCTGGAGGACTCGGGGCCTCCTCCTGTCCCCAAAGAAACGGTGGACTCTTTCCTCGACCTGGCCAGAAACCTGTTTCCCTCCAATCTTGTGGTTGCAGCTTTCCGTACGTATGCAACCGATTATAAAGTCGTGACCCAGAACAGCAGCTCTGGAAATGTAACCCATGAAAAGATCCCCATAGGCACTGAGATAGAAGGGATGAACATTTTAGGATTGGTCCTGTTTGCTCTGGTGTTAGGAGTGGCCTTAAAGAAACTAGGCTCCGAAGGAGAGGACCTCATCCGTTTCTTCAATTCCCTCAACGAGGCGACGATGGTGCTGGTGTCCTGGATTATGTGGTACGCGTCTTCAGGCTAGGTGGAGGCTCAGTG", false} + fragment2 := Part{"GAAGTGCCATTCCGCCTGACCTGAAGACCAGTACGTACCTGTGGGCATCATGTTCCTTGTTGGAAGCAAGATCGTGGAAATGAAAGACATCATCGTGCTGGTGACCAGCCTGGGGAAATACATCTTCGCATCTATATTGGGCCACGTCATTCATGGTGGTATCGTCCTGCCGCTGATTTATTTTGTTTTCACACGAAAAAACCCATTCAGATTCCTCCTGGGCCTCCTCGCCCCATTTGCGACAGCATTTGCTACGTGCTCCAGCTCAGCGACCCTTCCCTCTATGATGAAGTGCATTGAAGAGAACAATGGTGTGGACAAGAGGATCTCCAGGTTTATTCTCCCCATCGGGGCCACCGTGAACATGGACGGAGCAGCCATCTTCCAGTGTGTGGCCGCGGTGTTCATTGCGCAACTCAACAACGTAGAGCTCAACGCAGGACAGATTTTCACCATTCTAGTGACTGCCACAGCGTCCAGTGTTGGAGCAGCAGGCGTGCCAGCTGGAGGGGTCCTCACCATTGCCATTATCCTGGAGGCCATTGGGCTGCCTACTCATGATCTGCCTCTGATCCTGGCTGTGGACTGGATTGTGGACCGGACCACCACGGTGGTGAATGTGGAAGGGGATGCCCTGGGTGCAGGCATTCTCCACCACCTGAATCAGAAGGCAACAAAGAAAGGCGAGCAGGAACTTGCTGAGGTGAAAGTGGAAGCCATCCCCAACTGCAAGTCTGAGGAGGAAACCTCGCCCCTGGTGACACACCAGAACCCCGCTGGCCCCGTGGCCAGTGCCCCAGAACTGGAATCCAAGGAGTCGGTTCTGTGAAGAGCTTAGAGACCGACGACTGCCTAAGGACATTCGCTGCGTCTTCAGGCTAGGTGGAGGCTCAGTG", false} + + benchmarkGoldenGate(b, enzymeManager, []Part{fragment1, fragment2, popen}) +} diff --git a/clone/example_test.go b/clone/example_test.go new file mode 100644 index 000000000..85d12dc60 --- /dev/null +++ b/clone/example_test.go @@ -0,0 +1,31 @@ +package clone_test + +import ( + "fmt" + "log" + + "github.com/TimothyStiles/poly/clone" + "github.com/TimothyStiles/poly/seqhash" +) + +func ExampleGoldenGate() { + enzymeManager := clone.NewEnzymeManager(clone.GetBaseRestrictionEnzymes()) + // Fragment 1 has a palindrome at its start. This isn't very common but + // can occur. These two fragments are real DNA fragments used in the + // FreeGenes Project. They are used because they were on my computer + // - Keoni + fragment1 := clone.Part{"GAAGTGCCATTCCGCCTGACCTGAAGACCAGGAGAAACACGTGGCAAACATTCCGGTCTCAAATGGAAAAGAGCAACGAAACCAACGGCTACCTTGACAGCGCTCAAGCCGGCCCTGCAGCTGGCCCGGGCGCTCCGGGTACCGCCGCGGGTCGTGCACGTCGTTGCGCGGGCTTCCTGCGGCGCCAAGCGCTGGTGCTGCTCACGGTGTCTGGTGTTCTGGCAGGCGCCGGTTTGGGCGCGGCACTGCGTGGGCTCAGCCTGAGCCGCACCCAGGTCACCTACCTGGCCTTCCCCGGCGAGATGCTGCTCCGCATGCTGCGCATGATCATCCTGCCGCTGGTGGTCTGCAGCCTGGTGTCGGGCGCCGCCTCCCTCGATGCCAGCTGCCTCGGGCGTCTGGGCGGTATCGCTGTCGCCTACTTTGGCCTCACCACACTGAGTGCCTCGGCGCTCGCCGTGGCCTTGGCGTTCATCATCAAGCCAGGATCCGGTGCGCAGACCCTTCAGTCCAGCGACCTGGGGCTGGAGGACTCGGGGCCTCCTCCTGTCCCCAAAGAAACGGTGGACTCTTTCCTCGACCTGGCCAGAAACCTGTTTCCCTCCAATCTTGTGGTTGCAGCTTTCCGTACGTATGCAACCGATTATAAAGTCGTGACCCAGAACAGCAGCTCTGGAAATGTAACCCATGAAAAGATCCCCATAGGCACTGAGATAGAAGGGATGAACATTTTAGGATTGGTCCTGTTTGCTCTGGTGTTAGGAGTGGCCTTAAAGAAACTAGGCTCCGAAGGAGAGGACCTCATCCGTTTCTTCAATTCCCTCAACGAGGCGACGATGGTGCTGGTGTCCTGGATTATGTGGTACGCGTCTTCAGGCTAGGTGGAGGCTCAGTG", false} + fragment2 := clone.Part{"GAAGTGCCATTCCGCCTGACCTGAAGACCAGTACGTACCTGTGGGCATCATGTTCCTTGTTGGAAGCAAGATCGTGGAAATGAAAGACATCATCGTGCTGGTGACCAGCCTGGGGAAATACATCTTCGCATCTATATTGGGCCACGTCATTCATGGTGGTATCGTCCTGCCGCTGATTTATTTTGTTTTCACACGAAAAAACCCATTCAGATTCCTCCTGGGCCTCCTCGCCCCATTTGCGACAGCATTTGCTACGTGCTCCAGCTCAGCGACCCTTCCCTCTATGATGAAGTGCATTGAAGAGAACAATGGTGTGGACAAGAGGATCTCCAGGTTTATTCTCCCCATCGGGGCCACCGTGAACATGGACGGAGCAGCCATCTTCCAGTGTGTGGCCGCGGTGTTCATTGCGCAACTCAACAACGTAGAGCTCAACGCAGGACAGATTTTCACCATTCTAGTGACTGCCACAGCGTCCAGTGTTGGAGCAGCAGGCGTGCCAGCTGGAGGGGTCCTCACCATTGCCATTATCCTGGAGGCCATTGGGCTGCCTACTCATGATCTGCCTCTGATCCTGGCTGTGGACTGGATTGTGGACCGGACCACCACGGTGGTGAATGTGGAAGGGGATGCCCTGGGTGCAGGCATTCTCCACCACCTGAATCAGAAGGCAACAAAGAAAGGCGAGCAGGAACTTGCTGAGGTGAAAGTGGAAGCCATCCCCAACTGCAAGTCTGAGGAGGAAACCTCGCCCCTGGTGACACACCAGAACCCCGCTGGCCCCGTGGCCAGTGCCCCAGAACTGGAATCCAAGGAGTCGGTTCTGTGAAGAGCTTAGAGACCGACGACTGCCTAAGGACATTCGCTGCGTCTTCAGGCTAGGTGGAGGCTCAGTG", false} + + // pOpen plasmid series (https://stanford.freegenes.org/collections/open-genes/products/open-plasmids#description). I use it for essentially all my cloning. -Keoni + var popen = clone.Part{"TAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGGCCTACTATTAGCAACAACGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACCTGCACCAGTCAGTAAAACGACGGCCAGTAGTCAAAAGCCTCCGACCGGAGGCTTTTGACTTGGTTCAGGTGGAGTGGGAGTAgtcttcGCcatcgCtACTAAAagccagataacagtatgcgtatttgcgcgctgatttttgcggtataagaatatatactgatatgtatacccgaagtatgtcaaaaagaggtatgctatgaagcagcgtattacagtgacagttgacagcgacagctatcagttgctcaaggcatatatgatgtcaatatctccggtctggtaagcacaaccatgcagaatgaagcccgtcgtctgcgtgccgaacgctggaaagcggaaaatcaggaagggatggctgaggtcgcccggtttattgaaatgaacggctcttttgctgacgagaacagggGCTGGTGAAATGCAGTTTAAGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGTGATATTATTGACACGCCCGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCTGCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAAGCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAAGAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGATGTTCTGGGGAATATAAATGTCAGGCTCCCTTATACACAGgcgatgttgaagaccaCGCTGAGGTGTCAATCGTCGGAGCCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCATGGTCATAGCTGTTTCCTGAGAGCTTGGCAGGTGATGACACACATTAACAAATTTCGTGAGGAGTCTCCAGAAGAATGCCATTAATTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGG", true} + + bbsI, err := enzymeManager.GetEnzymeByName("BbsI") + if err != nil { + log.Fatalf("Something went wrong when trying to get the enzyme. Got error: %s", err) + } + Clones, _ := clone.GoldenGate([]clone.Part{fragment1, fragment2, popen}, bbsI) + + fmt.Println(seqhash.RotateSequence(Clones[0])) + // Output: AAAAAAAGGATCTCAAGAAGGCCTACTATTAGCAACAACGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACCTGCACCAGTCAGTAAAACGACGGCCAGTAGTCAAAAGCCTCCGACCGGAGGCTTTTGACTTGGTTCAGGTGGAGTGGGAGAAACACGTGGCAAACATTCCGGTCTCAAATGGAAAAGAGCAACGAAACCAACGGCTACCTTGACAGCGCTCAAGCCGGCCCTGCAGCTGGCCCGGGCGCTCCGGGTACCGCCGCGGGTCGTGCACGTCGTTGCGCGGGCTTCCTGCGGCGCCAAGCGCTGGTGCTGCTCACGGTGTCTGGTGTTCTGGCAGGCGCCGGTTTGGGCGCGGCACTGCGTGGGCTCAGCCTGAGCCGCACCCAGGTCACCTACCTGGCCTTCCCCGGCGAGATGCTGCTCCGCATGCTGCGCATGATCATCCTGCCGCTGGTGGTCTGCAGCCTGGTGTCGGGCGCCGCCTCCCTCGATGCCAGCTGCCTCGGGCGTCTGGGCGGTATCGCTGTCGCCTACTTTGGCCTCACCACACTGAGTGCCTCGGCGCTCGCCGTGGCCTTGGCGTTCATCATCAAGCCAGGATCCGGTGCGCAGACCCTTCAGTCCAGCGACCTGGGGCTGGAGGACTCGGGGCCTCCTCCTGTCCCCAAAGAAACGGTGGACTCTTTCCTCGACCTGGCCAGAAACCTGTTTCCCTCCAATCTTGTGGTTGCAGCTTTCCGTACGTATGCAACCGATTATAAAGTCGTGACCCAGAACAGCAGCTCTGGAAATGTAACCCATGAAAAGATCCCCATAGGCACTGAGATAGAAGGGATGAACATTTTAGGATTGGTCCTGTTTGCTCTGGTGTTAGGAGTGGCCTTAAAGAAACTAGGCTCCGAAGGAGAGGACCTCATCCGTTTCTTCAATTCCCTCAACGAGGCGACGATGGTGCTGGTGTCCTGGATTATGTGGTACGTACCTGTGGGCATCATGTTCCTTGTTGGAAGCAAGATCGTGGAAATGAAAGACATCATCGTGCTGGTGACCAGCCTGGGGAAATACATCTTCGCATCTATATTGGGCCACGTCATTCATGGTGGTATCGTCCTGCCGCTGATTTATTTTGTTTTCACACGAAAAAACCCATTCAGATTCCTCCTGGGCCTCCTCGCCCCATTTGCGACAGCATTTGCTACGTGCTCCAGCTCAGCGACCCTTCCCTCTATGATGAAGTGCATTGAAGAGAACAATGGTGTGGACAAGAGGATCTCCAGGTTTATTCTCCCCATCGGGGCCACCGTGAACATGGACGGAGCAGCCATCTTCCAGTGTGTGGCCGCGGTGTTCATTGCGCAACTCAACAACGTAGAGCTCAACGCAGGACAGATTTTCACCATTCTAGTGACTGCCACAGCGTCCAGTGTTGGAGCAGCAGGCGTGCCAGCTGGAGGGGTCCTCACCATTGCCATTATCCTGGAGGCCATTGGGCTGCCTACTCATGATCTGCCTCTGATCCTGGCTGTGGACTGGATTGTGGACCGGACCACCACGGTGGTGAATGTGGAAGGGGATGCCCTGGGTGCAGGCATTCTCCACCACCTGAATCAGAAGGCAACAAAGAAAGGCGAGCAGGAACTTGCTGAGGTGAAAGTGGAAGCCATCCCCAACTGCAAGTCTGAGGAGGAAACCTCGCCCCTGGTGACACACCAGAACCCCGCTGGCCCCGTGGCCAGTGCCCCAGAACTGGAATCCAAGGAGTCGGTTCTGTGAAGAGCTTAGAGACCGACGACTGCCTAAGGACATTCGCTGAGGTGTCAATCGTCGGAGCCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCATGGTCATAGCTGTTTCCTGAGAGCTTGGCAGGTGATGACACACATTAACAAATTTCGTGAGGAGTCTCCAGAAGAATGCCATTAATTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAG +}