dnaudit - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

This document describes documentation of genetic constructs in a light plain text format. The goal is to produce documentation that is clear and unambiguous, yet with minimal markup.

The documentation should show step-by step how a genetic construct was made so that it can be reproduced while at the same time making it easier to preserve and share genetic material.

A genetic construct can be for example:

a plasmid made from parts of other plasmids or chromosomal DNA fragments
DNA integrated in the genome of an organism by CRISPr

The documentation for a genetic construct consists of a a collection of text files in a folder 📁 with optional sub folders. Each text file contains one or more molecular biology unit-operations consisting of sequences in FASTA of Genbank format.

A very reduced, easy to remember collection of key words describe each unit-operation. Examples of unit operation are for
example PCR and homologous recombination.

A unit operation is delineated by a header (Table #1) and the next header or the end of the text file.

A unit operation contains a series of input sequences in a specified order as well as at least one resulting sequence. The file can (and should) also contain comments explaining the aim of the experiment.

Table#1	Header	Unit operation
	`# pcr`	PCR reaction
	`# cut`	Restriction digestion
	`# ligate`	Ligation with a DNA ligase
	`# homologous_recombination`	Homologous recombination
	`# crispr`	CRISPr cut
	`# fusion_pcr`	fusion PCR

🔑 Additionally, there are some reserved key/value expressions (Table#2) with special meaning.

🚨Key/Value Pairs should contain no white space characters.

Table#2	Reserved Key/Value Pairs	Meaning
	`cdseguid=...`	Checksum for a circular dsDNA sequence
	`ldseguid=...`	Checksum for a linear dsDNA sequence
	`csseguid=...`	Checksum for a circular ssDNA sequence
	`lsseguid=...`	Checksum for a linear ssDNA sequence
	`format=dsIUPAC`
	`topology=circular/linear`
	`molecule=protein/DNA/RNA`	Indicating a protein sequence.

These reserved Key/Value should be placed after the identifier in the FASTA header or in the comment section of a Genbank file. Restriction enzyme names such as BamHI or HindIII must be written as they appear in rebase.

Convention

Sequences must be in either FASTA or Genbank format.
All relevant files have to be in the same folder tree (project folder).
Cloning is described as a collection of unit-operations distributed in plain text files (.txt) or markdown (.md).
A unit-operation starts with one of the reserved words from Table#1 like for example # pcr.
Several unit-operation can be collected in one text file.
Sequences are identified by their name or identifier (This is not necessarily the same as the file name).
Identifiers and file names must be unique in the project folder tree.
There can not be more than one identifier for a certain DNA sequence in the project folder.
A DNA sequence can be followed by one ore more protein sequences, indicating that they are expected to be expressed from that DNA sequence.

The PydnaWeb simulation tools can be helpful when preparing strategy snippets.

Examples

pcr

# pcr

This snippet (the following five FASTA sequences) describes a PCR reaction.
This text are comments and can be added as long as no line starts with
or anything that can be parsed as a Genbank file.
The xyz1 and abc2 primers were used to amplify
a fragment from the qwerty1 template. The resulting product is pwpwq3
List the two primers first followed by the template and the PCR product last.
Optionally, add a protein sequence labeled "protein".
The order is important.
It is important to leave a blank line between sequences and comments and sequences.

Four sequences + protein

>xyz1 (a primer)
gatcGGATCCATGAACTCATATCACATTTGC

>abc2 (another primer)
gatAGATCCTTATGTTGAGTGTCTAGGG

>qwerty1 ldseguid=rqAbbXf_4MV3Pvg3WXQKlxPUgEA (the template)
ATGAACTCATATCACATTTGCTTCAACGACTGCCGCCTTCGCTGTATCCCTAGACACTCAACATAA

>pwpwq3 ldseguid=l8OpUk_5XHWeEKoduaByO-ZRcbs (the resulting product)
gatcGGATCCATGAACTCATATCACATTTGCTTCAACGACTGCCGCCTTCGCTGTATCCCTAGACACTCAACATAAGGATCTatc

>myprot protein=true (this is optionally a protein expressed from the sequence immediately above)
MNSYHICFNDCRLRCIPRHST*



# pcr

This is a PCR on a circular template. Inspection of the primers would show that the first primer
anneals after the second primer on the template below (`pUCmu_2019`). If the template was linear, no
PCR product would be formed.

>1780_pUCmu_bb_F
tagaaaagatcaaaggatcttc

>1779_pUCmu_bb_R
actcttcctttttcaatattattg

>pUCmu_2019 template circular
acgcgtcgcgaggccatatgggttaacccatggccaagcttgcatgcctgcaggtcgactctagaggatcccgggtaccgagctcgaattcggatatcctcgagactagtgggcccgtttaaacacatgtgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctactaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacata

>808bp_PCR_prod
actcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacataacgcgtcgcgaggccatatgggttaacccatggccaagcttgcatgcctgcaggtcgactctagaggatcccgggtaccgagctcgaattcggatatcctcgagactagtgggcccgtttaaacacatgtgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttcta



# pcr

This PCR amplifies a an expression cassette containing the KanMX4 marker.

>1778_kan_F
atgcttcaataatattgaaaaaggaagagtATGGGTAAGGAAAAGACT

>1777_kan_R
ctcaagaagatcctttgatcttttctaTTAGAAAAACTCATCGAGCAT

>pFA6a_GFPS65T_kanMX6 template
gaacgcggccgccagctgaagcttcgtacgctgcaggtcgacggatccccgggttaattaacagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcacttatggtgttcaatgcttttcaagatacccagatcatatgaaacggcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaatagggcgcgccacttctaaataagcgaatttcttatgatttatgatttttattattaaataagttataaaaaaaataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattcttattcttgagtaactctttcctgtaggtcaggttgctttctcaggtatagtatgaggtcgctcttattgaccacacctctaccggcagatccgctagggataacagggtaatatagatctgtttagcttgcctcgtccccgccgggtcacccggccagcgacatggaggcccagaataccctccttgacagtcttgacgtgcgcagctcaggggcatgatgtgactgtcgcccgtacatttagcccatacatccccatgtataatcatttgcatccatacattttgatggccgcacggcgcgaagcaaaaattacggctcctcgctgcagacctgcgagcagggaaacgctcccctcacagacgcgttgaattgtccccacgccgcgcccctgtagagaaatataaaaggttaggatttgccactgaggttcttctttcatatacttccttttaaaatcttgctaggatacagttctcacatcacatccgaacataaacaaccatgggtaaggaaaagactcacgtttcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccggcaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatcagtactgacaataaaaagattcttgttttcaagaacttgtcatttgtatagtttttttatattgtagttgttctattttaatcaaatgttagcgtgatttatattttttttcgcctcgacatcatctgcccagatgcgaagttaagtgcgcagaaagtaatatcatgcgtcaatcgtatgtgaatgctggtcgctatactgctgtcgattcgatactaacgccgccatccagtttaaacgagctcgaattcatcgatgatatcagatccactagtggcctatgcggccgcggatctgccggtctccctatagtgagtcgtattaatttcgataagccaggttaacctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatggacatattgtcgttagaacgcggctacaattaatacataaccttatgtatcatacacatacgatttaggtgacactata

>867bp_PCR_prod
atgcttcaataatattgaaaaaggaagagtatgggtaaggaaaagactcacgtttcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccggcaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatagaaaagatcaaaggatcttcttgag



# pcr

This element describe a PCR product for in a yeast gene knockout cassette.

>1776_rv_ERG10_KanMX_del
TGTATTTTATGAAAAAGATCATGAGAAAATCGCAGAACGTAATCAgcataggccactagtggatctg

>1775_fw_ERG10_KanMX_del
GCGCCATATCATATATATTTATACAGATTAGACGTACTCAAAATGcagctgaagcttcgtacgc

LOCUS       AJ002680                3941 bp    DNA     linear   SYN 01-SEP-2004
DEFINITION  Synthetic DNA containing Escherichia coli plasmid pFA6a-kanMX4,
            kanamycin and geneticin (G418) resistance gene, bla gene, multiple
            cloning site, pSP72 derivative.
            template
ACCESSION   AJ002680
VERSION     AJ002680.1  GI:2623975
KEYWORDS    .
SOURCE      synthetic construct
  ORGANISM  synthetic construct
            other sequences; artificial sequences.
REFERENCE   1
  AUTHORS   Oka,A., Sugisaki,H. and Takanami,M.
  TITLE     Nucleotide sequence of the kanamycin resistance transposon Tn903
  JOURNAL   J. Mol. Biol. 147 (2), 217-226 (1981)
   PUBMED   6270337
REFERENCE   2
  AUTHORS   Steiner,S. and Philippsen,P.
  TITLE     Sequence and promoter analysis of the highly expressed TEF gene of
            the filamentous fungus Ashbya gossypii
  JOURNAL   Mol. Gen. Genet. 242 (3), 263-271 (1994)
   PUBMED   8107673
REFERENCE   3
  AUTHORS   Wach,A., Brachat,A., Pohlmann,R. and Philippsen,P.
  TITLE     New heterologous modules for classical or PCR-based gene
            disruptions in Saccharomyces cerevisiae
  JOURNAL   Yeast 10 (13), 1793-1808 (1994)
   PUBMED   7747518
REFERENCE   4  (bases 1 to 3941)
  AUTHORS   Philippsen,P.
  TITLE     Direct Submission
  JOURNAL   Submitted (07-NOV-1997) Philippsen P., Applied Microbiology,
            University of Basel, Biozentrum, Klingelbergstr. 70, Basel,
            CH-4056, SWITZERLAND
FEATURES             Location/Qualifiers
     source          1..3941
                     /organism="synthetic construct"
                     /mol_type="other DNA"
                     /db_xref="taxon:32630"
                     /focus
     source          80..482
                     /organism="Eremothecium gossypii"
                     /mol_type="other DNA"
                     /db_xref="taxon:33169"
     source          483..1271
                     /organism="Escherichia coli"
                     /mol_type="other DNA"
                     /db_xref="taxon:562"
     source          1277..1501
                     /organism="Eremothecium gossypii"
                     /mol_type="other DNA"
                     /db_xref="taxon:33169"
     misc_feature    1..79
                     /note="artificial sequence"
     misc_feature    5..75
                     /note="multiple cloning site"
                     /citation=[3]
     gene            115..458
                     /gene="TEF"
     promoter        115..458
                     /gene="TEF"
                     /note="Translation elongation factor 1 alpha"
                     /citation=[2]
     CDS             459..1268
                     /note="unnamed protein product; Kanamycin resistance gene"
                     /citation=[1]
                     /codon_start=1
                     /transl_table=11
                     /protein_id="CAA05683.1"
                     /db_xref="GI:2623976"
                     /db_xref="InterPro:IPR002575"
                     /db_xref="InterPro:IPR008266"
                     /db_xref="InterPro:IPR011009"
                     /db_xref="InterPro:IPR012149"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGK
                     PDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVD
                     ASDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRV
                     GIADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     repeat_region   483..1271
                     /mobile_element="transposon:Tn903"
     misc_feature    1272..1276
                     /note="artificial sequence"
     gene            1277..1471
                     /gene="TEF"
     terminator      1277..1471
                     /gene="TEF"
                     /note="translation elongation factor 1 alpha"
                     /citation=[2]
     misc_feature    1502..3941
                     /note="artificial sequence"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acggatcccc gggttaatta
       61 aggcgcgcca gatctgttta gcttgcctcg tccccgccgg gtcacccggc cagcgacatg
      121 gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac
      181 tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc catacatttt
      241 gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcagacctg cgagcaggga
      301 aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata
      361 aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta
      421 ggatacagtt ctcacatcac atccgaacat aaacaaccat gggtaaggaa aagactcacg
      481 tttcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc
      541 gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc
      601 cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg
      661 tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta
      721 ctcctgatga tgcatggtta ctcaccactg cgatccccgg caaaacagca ttccaggtat
      781 tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc
      841 ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg
      901 ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc
      961 gtaatggctg gcctgttgaa caagtctgga aagaaatgca taagcttttg ccattctcac
     1021 cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga
     1081 aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg
     1141 ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa
     1201 aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt
     1261 ttttctaatc agtactgaca ataaaaagat tcttgttttc aagaacttgt catttgtata
     1321 gtttttttat attgtagttg ttctatttta atcaaatgtt agcgtgattt atattttttt
     1381 tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa tatcatgcgt
     1441 caatcgtatg tgaatgctgg tcgctatact gctgtcgatt cgatactaac gccgccatcc
     1501 agtgtcgaaa acgagctcga attcatcgat gatatcagat ccactagtgg cctatgcggc
     1561 cgcggatctg ccggtctccc tatagtgagt cgtattaatt tcgataagcc aggttaacct
     1621 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     1681 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     1741 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     1801 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     1861 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     1921 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     1981 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     2041 gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     2101 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     2161 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     2221 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     2281 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     2341 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     2401 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     2461 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     2521 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     2581 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     2641 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     2701 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     2761 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     2821 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     2881 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     2941 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3001 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3061 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     3121 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     3181 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     3241 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     3301 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     3361 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     3421 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     3481 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     3541 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     3601 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     3661 gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     3721 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     3781 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
     3841 tgtactgaga gtgcaccata tggacatatt gtcgttagaa cgcggctaca attaatacat
     3901 aaccttatgt atcatacaca tacgatttag gtgacactat a
//

>1635bp_PCR_prod (this is the cassette)
GCGCCATATCATATATATTTATACAGATTAGACGTACTCAAAATGCAGCTGAAGCTTCGTACGCTGCAGGTCGACGGATCCCCGGGTTAATTAAGGCGCGCCAGATCTGTTTAGCTTGCCTCGTCCCCGCCGGGTCACCCGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCTCCCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAACCATGGGTAAGGAAAAGACTCACGTTTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGTACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGAAAACGAGCTCGAATTCATCGATGATATCAGATCCACTAGTGGCCTATGCTGATTACGTTCTGCGATTTTCTCATGATCTTTTTCATAAAATACA

ligation

# ligate

This snippet describes a cut&ligate cloning procedure. The pwpwq3 PCR product is cloned in the EcoRV site of vector pUCmuK.
This is a blunt cloning and the PCR product is *not* digested with any restriction enzyme.
Restriction enzymes has to  
Sequence immediately following the restriction enzyme.
It is important to note that circular topology for FASTA sequences are indicated by `circular` in the header line.

>pwpwq3 ldseguid=l8OpUk_5XHWeEKoduaByO-ZRcbs (this is the insert, linear dsDNA)
gatcGGATCCATGAACTCATATCACATTTGCTTCAACGACTGCCGCCTTCGCTGTATCCCTAGACACTCAACATAAGGATCTatc

>pUCmuK cdseguid=r3Oi6LjFyOLB64eWO2Q-LeRtyq0 cut=EcoRV (this is a circular ds DNA vector) 
actcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacataacgcgtcgcgaggccatatgggttaacccatggccaagcttgcatgcctgcaggtcgactctagaggatcccgggtaccgagctcgaattcggatatcctcgagactagtgggcccgtttaaacacatgtgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttgccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgaaacgtgagtcttttccttacccat

>pUCmuK_pwpwq3 cdseguid=fk6zm54GgaVFAhbvA11wZxEaQ_8 (this is the resulting circular plasmid)
actcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacataacgcgtcgcgaggccatatgggttaacccatggccaagcttgcatgcctgcaggtcgactctagaggatcccgggtaccgagctcgaattcggatgatcGGATCCATGAACTCATATCACATTTGCTTCAACGACTGCCGCCTTCGCTGTATCCCTAGACACTCAACATAAGGATCTatcatcctcgagactagtgggcccgtttaaacacatgtgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttgccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgaaacgtgagtcttttccttacccat

>myp protein=true (this is optionally a protein expressed from the sequence immediately above)
MNSYHICFNDCRLRCIPRHST*



# ligate

This is a sticky end cloning. The pwpwq3 PCR product digested with BamHI & XhoII and
cloned in the BamHI site of vector pUCmuK.

>pwpwq3 linear ldseguid=O8pSBFI3EqOsXu9_a1235LCh9O8 (this is the insert) 
LEOFCATGAACTCATATCACATTTGCTTCAACGACTGCCGCCTTCGCTGTATCCCTAGACACTCAACATAQZPX

>pUCmuK circular (this is the vector) cdseguid=r3Oi6LjFyOLB64eWO2Q-LeRtyq0 cut=BamHI
actcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacataacgcgtcgcgaggccatatgggttaacccatggccaagcttgcatgcctgcaggtcgactctagaggatcccgggtaccgagctcgaattcggatatcctcgagactagtgggcccgtttaaacacatgtgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttgccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgaaacgtgagtcttttccttacccat

>pUCmuK_pwpwq3_sticky cdseguid=sdtvgK1IVf-Jtc6rzS0WJpLG4C0 (this is the resulting vector)
actcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacataacgcgtcgcgaggccatatgggttaacccatggccaagcttgcatgcctgcaggtcgactctagagGATCCATGAACTCATATCACATTTGCTTCAACGACTGCCGCCTTCGCTGTATCCCTAGACACTCAACATAAgatcccgggtaccgagctcgaattcggatatcctcgagactagtgggcccgtttaaacacatgtgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttgccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgaaacgtgagtcttttccttacccat

homologous recombination

# homologous_recombination

This element describes a recombination between the two previous linear PCR products forming a new circular plasmid.

 -|808bp_PCR_prod|27
|                 \/
|                 /\
|                 27|867bp_PCR_pro_rc|30
|                                     \/
|                                     /\
|                                     30-
|                                        |
 ----------------------------------------


>808bp_PCR_prod ldseguid=Dz3zSkRgSWMrtc0AGAZf-lFpbYQ (one DNA fragment)
actcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacataacgcgtcgcgaggccatatgggttaacccatggccaagcttgcatgcctgcaggtcgactctagaggatcccgggtaccgagctcgaattcggatatcctcgagactagtgggcccgtttaaacacatgtgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttcta

>867bp_PCR_prod ldseguid=8cjo-NQlkgLkuRlwS1qT-N10OTk (another DNA fragment)
atgcttcaataatattgaaaaaggaagagtatgggtaaggaaaagactcacgtttcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccggcaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaatagaaaagatcaaaggatcttcttgag

>pUCmuK cdseguid=r3Oi6LjFyOLB64eWO2Q-LeRtyq0 (the resulting final plasmid )
actcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacataacgcgtcgcgaggccatatgggttaacccatggccaagcttgcatgcctgcaggtcgactctagaggatcccgggtaccgagctcgaattcggatatcctcgagactagtgggcccgtttaaacacatgtgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttgccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgaaacgtgagtcttttccttacccat




# homologous_recombination

This snippet describes the recombination between the ERG10 locus and the cassette.
Note that the locus sequence is repeated. This is necessary for technical reasons and has to do with how the
internals of the Pydna Assembly class.

ERG10_locus|45
            \/
            /\
            45|1635bp_PCR_prod|45
                               \/
                               /\
                               45|ERG10_locus

>ERG10_locus lSEGUID
ATTGAAGCACCTGTGGAGTATTTAAAAACTGCGGTTACATGGCCTACAGATGAAATATGTGCTCAACTAATGACACAATTCCCACCAGGAACGCCGACCAGTGTCCTGCTGCAGACTATTTCAGATGAGCTAGAGAAAAGTTCTGACAACCTGTTCACGTTATCTGATTTAAAGAGCAAACTGAAAGTTATTGGCTTATTCGAGCACATGGAAGATATCCCATTTTTCGACAAGCTGAAACTAAGCAATGCGCCCGTGAAGGACATGCCTATGGTCACAAAGGCGTTCACCAAATTTTGCGAAACAATAGCAAAAAGGCATACAAGAGGCCTACTGTCATACCGATTACCTTTTAACCTACTGGACTACAATTGCATACCGAATGAGAGTTATTCATTAGAGGTTTATGAGTCATTGTACAACATCATTACTCTATACTTCTGGCTCAGCAACAGGTACCCAAACTACTTCATTGACATGGAATCTGCTAAAGATTTGAAGTATTTCTGTGAGATGATTATTTTCGAGAAACTTGATCGATTAAAGAAGAATCCTTACGCACATAAGCCCTTTGGTTCTACAAGAGGTCACCTCTCATCTTCGAGAAGAAGATTGCGTACATAATCTACGATATATCCTGTAAATAGAAACAGCTACACTGCTTGAAAGCCTTAACATGATACATTTCTGGTATGATGCCATTGTTGTGCCCTGCCGGGTTTATCGTTTCCTAACAGGCACGTCACTTATAACGAGGTGCCTGTCGTTTACCGCCCAAGCCGGTTTTTTCGCTGGAGAGTACGGTACTACTAGCCCACCACACGTTCGTGGCCAGGTTGATAGGCCACCGTTGAGCAAAGGGCAGTAAAATATATAAAAGAGGAACAAGCGCTTCCATTAAGAGCACTGCTAAGCCTACTCGTTTTCTAGTTCTCTGAAAAAAGGTAGCCTAAAACAAGCGCCATATCATATATATTTATACAGATTAGACGTACTCAAAATGTCTCAGAACGTTTACATTGTATCGACTGCCAGAACCCCAATTGGTTCATTCCAGGGTTCTCTATCCTCCAAGACAGCAGTGGAATTGGGTGCTGTTGCTTTAAAAGGCGCCTTGGCTAAGGTTCCAGAATTGGATGCATCCAAGGATTTTGACGAAATTATTTTTGGTAACGTTCTTTCTGCCAATTTGGGCCAAGCTCCGGCCAGACAAGTTGCTTTGGCTGCCGGTTTGAGTAATCATATCGTTGCAAGCACAGTTAACAAGGTCTGTGCATCCGCTATGAAGGCAATCATTTTGGGTGCTCAATCCATCAAATGTGGTAATGCTGATGTTGTCGTAGCTGGTGGTTGTGAATCTATGACTAACGCACCATACTACATGCCAGCAGCCCGTGCGGGTGCCAAATTTGGCCAAACTGTTCTTGTTGATGGTGTCGAAAGAGATGGGTTGAACGATGCGTACGATGGTCTAGCCATGGGTGTACACGCAGAAAAGTGTGCCCGTGATTGGGATATTACTAGAGAACAACAAGACAATTTTGCCATCGAATCCTACCAAAAATCTCAAAAATCTCAAAAGGAAGGTAAATTCGACAATGAAATTGTACCTGTTACCATTAAGGGATTTAGAGGTAAGCCTGATACTCAAGTCACGAAGGACGAGGAACCTGCTAGATTACACGTTGAAAAATTGAGATCTGCAAGGACTGTTTTCCAAAAAGAAAACGGTACTGTTACTGCCGCTAACGCTTCTCCAATCAACGATGGTGCTGCAGCCGTCATCTTGGTTTCCGAAAAAGTTTTGAAGGAAAAGAATTTGAAGCCTTTGGCTATTATCAAAGGTTGGGGTGAGGCCGCTCATCAACCAGCTGATTTTACATGGGCTCCATCTCTTGCAGTTCCAAAGGCTTTGAAACATGCTGGCATCGAAGACATCAATTCTGTTGATTACTTTGAATTCAATGAAGCCTTTTCGGTTGTCGGTTTGGTGAACACTAAGATTTTGAAGCTAGACCCATCTAAGGTTAATGTATATGGTGGTGCTGTTGCTCTAGGTCACCCATTGGGTTGTTCTGGTGCTAGAGTGGTTGTTACACTGCTATCCATCTTACAGCAAGAAGGAGGTAAGATCGGTGTTGCCGCCATTTGTAATGGTGGTGGTGGTGCTTCCTCTATTGTCATTGAAAAGATATGATTACGTTCTGCGATTTTCTCATGATCTTTTTCATAAAATACATAAATATATAAATGGCTTTATGTATAACAGGCATAATTTAAAGTTTTATTTGCGATTCATCGTTTTTCAGGTACTCAAACGCTGAGGTGTGCCTTTTGACTTACTTTTCCGCCTTGGCAAGCTGGCCGGGTGATACTTGCACAAGTTCCACTAATTACTGACATTTGTGGTATTAACTCGTTTGACTGCTCTACAATTGTAGGATGTTAATCAATGTCTTGGCTGCCTTCATTCTCTTCAGGCTCTATTAATTTTAACCGTTATAAGTTCCTTTTCTCCCTTGGAAGCAAACATCAACTGCCTTAAAATCTGGTGGCGAGGAAAGAGGAAATGGCATGTACTAATGATGGTCCTAATAAATATCCCGAAATTGTGAGTGTTAAGCACCTGTTCCAACATTCGGGATCCAAGCATGAATTTAGTGCTGGTAAACGATTTTCAAAATCCATTGGTAAAATATTCAAACGAAACTCTGCTTTGAAAACTTCTAGAACTGAAACGGCAAATCATAAAATGGAATTGAAAAAAAGAGAGGGTGTTACCTTATTGCCACCTGTCCCAGAATCATTATTACATAAACTCAATTCTTGGTTGGAAACTTTTTCTTCCACCAAGAACATGAAAATCGAAGAAAACAAAATTGTTATTAATGAAAAAGAGATTCGGGATTCAGTCTCTTACTACCCTGATAAGAATGGAGGAAGTGCTGTATTTTGTTACTTGCCCGACCTTGTGCTATATTATAAGCCGCCTATAAAAGTCACAGGCAAGCAATGTCCAATAAAGAGAAGTCCTTGGGAATCGATGGAAATCCAATATCAAAAGTTTATGTACCCCTTAGAAAGGTTGGAAAGACAGTTTGAGGAAGTTCCATTTAGGCCCTGGTATTTTGCAATGCGATTAAAGGAACTTTACAGATGCTGTGAAAGGTCTTTTACTAACGCGGCAAATAGAGGAA

>1635bp_PCR_prod lSEGUID
GCGCCATATCATATATATTTATACAGATTAGACGTACTCAAAATGCAGCTGAAGCTTCGTACGCTGCAGGTCGACGGATCCCCGGGTTAATTAAGGCGCGCCAGATCTGTTTAGCTTGCCTCGTCCCCGCCGGGTCACCCGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCTCCCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAACCATGGGTAAGGAAAAGACTCACGTTTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGTACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGAAAACGAGCTCGAATTCATCGATGATATCAGATCCACTAGTGGCCTATGCTGATTACGTTCTGCGATTTTCTCATGATCTTTTTCATAAAATACA

>ERG10_locus lSEGUID
ATTGAAGCACCTGTGGAGTATTTAAAAACTGCGGTTACATGGCCTACAGATGAAATATGTGCTCAACTAATGACACAATTCCCACCAGGAACGCCGACCAGTGTCCTGCTGCAGACTATTTCAGATGAGCTAGAGAAAAGTTCTGACAACCTGTTCACGTTATCTGATTTAAAGAGCAAACTGAAAGTTATTGGCTTATTCGAGCACATGGAAGATATCCCATTTTTCGACAAGCTGAAACTAAGCAATGCGCCCGTGAAGGACATGCCTATGGTCACAAAGGCGTTCACCAAATTTTGCGAAACAATAGCAAAAAGGCATACAAGAGGCCTACTGTCATACCGATTACCTTTTAACCTACTGGACTACAATTGCATACCGAATGAGAGTTATTCATTAGAGGTTTATGAGTCATTGTACAACATCATTACTCTATACTTCTGGCTCAGCAACAGGTACCCAAACTACTTCATTGACATGGAATCTGCTAAAGATTTGAAGTATTTCTGTGAGATGATTATTTTCGAGAAACTTGATCGATTAAAGAAGAATCCTTACGCACATAAGCCCTTTGGTTCTACAAGAGGTCACCTCTCATCTTCGAGAAGAAGATTGCGTACATAATCTACGATATATCCTGTAAATAGAAACAGCTACACTGCTTGAAAGCCTTAACATGATACATTTCTGGTATGATGCCATTGTTGTGCCCTGCCGGGTTTATCGTTTCCTAACAGGCACGTCACTTATAACGAGGTGCCTGTCGTTTACCGCCCAAGCCGGTTTTTTCGCTGGAGAGTACGGTACTACTAGCCCACCACACGTTCGTGGCCAGGTTGATAGGCCACCGTTGAGCAAAGGGCAGTAAAATATATAAAAGAGGAACAAGCGCTTCCATTAAGAGCACTGCTAAGCCTACTCGTTTTCTAGTTCTCTGAAAAAAGGTAGCCTAAAACAAGCGCCATATCATATATATTTATACAGATTAGACGTACTCAAAATGTCTCAGAACGTTTACATTGTATCGACTGCCAGAACCCCAATTGGTTCATTCCAGGGTTCTCTATCCTCCAAGACAGCAGTGGAATTGGGTGCTGTTGCTTTAAAAGGCGCCTTGGCTAAGGTTCCAGAATTGGATGCATCCAAGGATTTTGACGAAATTATTTTTGGTAACGTTCTTTCTGCCAATTTGGGCCAAGCTCCGGCCAGACAAGTTGCTTTGGCTGCCGGTTTGAGTAATCATATCGTTGCAAGCACAGTTAACAAGGTCTGTGCATCCGCTATGAAGGCAATCATTTTGGGTGCTCAATCCATCAAATGTGGTAATGCTGATGTTGTCGTAGCTGGTGGTTGTGAATCTATGACTAACGCACCATACTACATGCCAGCAGCCCGTGCGGGTGCCAAATTTGGCCAAACTGTTCTTGTTGATGGTGTCGAAAGAGATGGGTTGAACGATGCGTACGATGGTCTAGCCATGGGTGTACACGCAGAAAAGTGTGCCCGTGATTGGGATATTACTAGAGAACAACAAGACAATTTTGCCATCGAATCCTACCAAAAATCTCAAAAATCTCAAAAGGAAGGTAAATTCGACAATGAAATTGTACCTGTTACCATTAAGGGATTTAGAGGTAAGCCTGATACTCAAGTCACGAAGGACGAGGAACCTGCTAGATTACACGTTGAAAAATTGAGATCTGCAAGGACTGTTTTCCAAAAAGAAAACGGTACTGTTACTGCCGCTAACGCTTCTCCAATCAACGATGGTGCTGCAGCCGTCATCTTGGTTTCCGAAAAAGTTTTGAAGGAAAAGAATTTGAAGCCTTTGGCTATTATCAAAGGTTGGGGTGAGGCCGCTCATCAACCAGCTGATTTTACATGGGCTCCATCTCTTGCAGTTCCAAAGGCTTTGAAACATGCTGGCATCGAAGACATCAATTCTGTTGATTACTTTGAATTCAATGAAGCCTTTTCGGTTGTCGGTTTGGTGAACACTAAGATTTTGAAGCTAGACCCATCTAAGGTTAATGTATATGGTGGTGCTGTTGCTCTAGGTCACCCATTGGGTTGTTCTGGTGCTAGAGTGGTTGTTACACTGCTATCCATCTTACAGCAAGAAGGAGGTAAGATCGGTGTTGCCGCCATTTGTAATGGTGGTGGTGGTGCTTCCTCTATTGTCATTGAAAAGATATGATTACGTTCTGCGATTTTCTCATGATCTTTTTCATAAAATACATAAATATATAAATGGCTTTATGTATAACAGGCATAATTTAAAGTTTTATTTGCGATTCATCGTTTTTCAGGTACTCAAACGCTGAGGTGTGCCTTTTGACTTACTTTTCCGCCTTGGCAAGCTGGCCGGGTGATACTTGCACAAGTTCCACTAATTACTGACATTTGTGGTATTAACTCGTTTGACTGCTCTACAATTGTAGGATGTTAATCAATGTCTTGGCTGCCTTCATTCTCTTCAGGCTCTATTAATTTTAACCGTTATAAGTTCCTTTTCTCCCTTGGAAGCAAACATCAACTGCCTTAAAATCTGGTGGCGAGGAAAGAGGAAATGGCATGTACTAATGATGGTCCTAATAAATATCCCGAAATTGTGAGTGTTAAGCACCTGTTCCAACATTCGGGATCCAAGCATGAATTTAGTGCTGGTAAACGATTTTCAAAATCCATTGGTAAAATATTCAAACGAAACTCTGCTTTGAAAACTTCTAGAACTGAAACGGCAAATCATAAAATGGAATTGAAAAAAAGAGAGGGTGTTACCTTATTGCCACCTGTCCCAGAATCATTATTACATAAACTCAATTCTTGGTTGGAAACTTTTTCTTCCACCAAGAACATGAAAATCGAAGAAAACAAAATTGTTATTAATGAAAAAGAGATTCGGGATTCAGTCTCTTACTACCCTGATAAGAATGGAGGAAGTGCTGTATTTTGTTACTTGCCCGACCTTGTGCTATATTATAAGCCGCCTATAAAAGTCACAGGCAAGCAATGTCCAATAAAGAGAAGTCCTTGGGAATCGATGGAAATCCAATATCAAAAGTTTATGTACCCCTTAGAAAGGTTGGAAAGACAGTTTGAGGAAGTTCCATTTAGGCCCTGGTATTTTGCAATGCGATTAAAGGAACTTTACAGATGCTGTGAAAGGTCTTTTACTAACGCGGCAAATAGAGGAA

>ERG10_locus_kanmx_ERG10_locus
ATTGAAGCACCTGTGGAGTATTTAAAAACTGCGGTTACATGGCCTACAGATGAAATATGT
GCTCAACTAATGACACAATTCCCACCAGGAACGCCGACCAGTGTCCTGCTGCAGACTATT
TCAGATGAGCTAGAGAAAAGTTCTGACAACCTGTTCACGTTATCTGATTTAAAGAGCAAA
CTGAAAGTTATTGGCTTATTCGAGCACATGGAAGATATCCCATTTTTCGACAAGCTGAAA
CTAAGCAATGCGCCCGTGAAGGACATGCCTATGGTCACAAAGGCGTTCACCAAATTTTGC
GAAACAATAGCAAAAAGGCATACAAGAGGCCTACTGTCATACCGATTACCTTTTAACCTA
CTGGACTACAATTGCATACCGAATGAGAGTTATTCATTAGAGGTTTATGAGTCATTGTAC
AACATCATTACTCTATACTTCTGGCTCAGCAACAGGTACCCAAACTACTTCATTGACATG
GAATCTGCTAAAGATTTGAAGTATTTCTGTGAGATGATTATTTTCGAGAAACTTGATCGA
TTAAAGAAGAATCCTTACGCACATAAGCCCTTTGGTTCTACAAGAGGTCACCTCTCATCT
TCGAGAAGAAGATTGCGTACATAATCTACGATATATCCTGTAAATAGAAACAGCTACACT
GCTTGAAAGCCTTAACATGATACATTTCTGGTATGATGCCATTGTTGTGCCCTGCCGGGT
TTATCGTTTCCTAACAGGCACGTCACTTATAACGAGGTGCCTGTCGTTTACCGCCCAAGC
CGGTTTTTTCGCTGGAGAGTACGGTACTACTAGCCCACCACACGTTCGTGGCCAGGTTGA
TAGGCCACCGTTGAGCAAAGGGCAGTAAAATATATAAAAGAGGAACAAGCGCTTCCATTA
AGAGCACTGCTAAGCCTACTCGTTTTCTAGTTCTCTGAAAAAAGGTAGCCTAAAACAAGC
GCCATATCATATATATTTATACAGATTAGACGTACTCAAAATGCAGCTGAAGCTTCGTAC
GCTGCAGGTCGACGGATCCCCGGGTTAATTAAGGCGCGCCAGATCTGTTTAGCTTGCCTC
GTCCCCGCCGGGTCACCCGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAGTCT
TGACGTGCGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATACATCC
CCATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGCGAAGCAAAAATTA
CGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCTCCCCTCACAGACGCGTTGAATTG
TCCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTAGGATTTGCCACTGAGGTTCTT
CTTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACA
TAAACAACCATGGGTAAGGAAAAGACTCACGTTTCGAGGCCGCGATTAAATTCCAACATG
GATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACA
ATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGT
AGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATG
CCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACT
GCGATCCCCGGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAAT
ATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGT
CCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGT
TTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGG
AAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTC
TCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGA
GTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTT
TCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAAT
AAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGTACTGACAATAAAAAGA
TTCTTGTTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTT
AATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGA
AGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATAC
TGCTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGAAAACGAGCTCGAATTCATCGA
TGATATCAGATCCACTAGTGGCCTATGCTGATTACGTTCTGCGATTTTCTCATGATCTTT
TTCATAAAATACATAAATATATAAATGGCTTTATGTATAACAGGCATAATTTAAAGTTTT
ATTTGCGATTCATCGTTTTTCAGGTACTCAAACGCTGAGGTGTGCCTTTTGACTTACTTT
TCCGCCTTGGCAAGCTGGCCGGGTGATACTTGCACAAGTTCCACTAATTACTGACATTTG
TGGTATTAACTCGTTTGACTGCTCTACAATTGTAGGATGTTAATCAATGTCTTGGCTGCC
TTCATTCTCTTCAGGCTCTATTAATTTTAACCGTTATAAGTTCCTTTTCTCCCTTGGAAG
CAAACATCAACTGCCTTAAAATCTGGTGGCGAGGAAAGAGGAAATGGCATGTACTAATGA
TGGTCCTAATAAATATCCCGAAATTGTGAGTGTTAAGCACCTGTTCCAACATTCGGGATC
CAAGCATGAATTTAGTGCTGGTAAACGATTTTCAAAATCCATTGGTAAAATATTCAAACG
AAACTCTGCTTTGAAAACTTCTAGAACTGAAACGGCAAATCATAAAATGGAATTGAAAAA
AAGAGAGGGTGTTACCTTATTGCCACCTGTCCCAGAATCATTATTACATAAACTCAATTC
TTGGTTGGAAACTTTTTCTTCCACCAAGAACATGAAAATCGAAGAAAACAAAATTGTTAT
TAATGAAAAAGAGATTCGGGATTCAGTCTCTTACTACCCTGATAAGAATGGAGGAAGTGC
TGTATTTTGTTACTTGCCCGACCTTGTGCTATATTATAAGCCGCCTATAAAAGTCACAGG
CAAGCAATGTCCAATAAAGAGAAGTCCTTGGGAATCGATGGAAATCCAATATCAAAAGTT
TATGTACCCCTTAGAAAGGTTGGAAAGACAGTTTGAGGAAGTTCCATTTAGGCCCTGGTA
TTTTGCAATGCGATTAAAGGAACTTTACAGATGCTGTGAAAGGTCTTTTACTAACGCGGC
AAATAGAGGAA

crispr

# crispr

This snippet describe a CRISPr digestion and recombination

>guide sgRNA construct, partial or whole plasmid (better)
gatc....

>donor dna (linear)
gatc

>target DNA to be cut (linear *or* circular)
gatc...

>sequence1 a result from recombination between donor and target, can be linear or circular
gatc...

>sequence2 another result from recombination between donor and target, can be linear or circular
actg...

...

How?

Look for the sgRNA constant parts (GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG) in guide construct which is the first sequence.
Extract the guide sequence, defining the cut (20 nt).
Find guide sequence in target, verify presence of PAM.
Cut the target DNA into a series of linear sequences.
Recombine donor and targets to one or more sequences
Indicate if guide still cuts the recombined sequence(s)

More examples

A lactose metabolic pathway for S. cerevisiae was formulated using this method. The pathway contains two expression cassettes with the Kluyveromyces lactis beta-D-galactosidase LAC4 and the LAC12 Kluyveromyces lactis LAC12 gene for lactose permease. You can download the files by using the Code button and then the Download ZIP button:

dlzip.png

Briefly:

LAC4 was pcr amplified from chromosomal DNA.
The PCR product was cloned in the pYPKa vector in the AjiI site.
A DNA fragment was pcr amplified from the pYPKa_A_LAC4 vector
A promoter was amplified from the pYPKa_Z_PDC1 plasmid
A terminator was amplified from the pYPKa_E_PGI1 plasmid
The promoter gene and terminator and the vector pYPKpw digested with ZraI was joined by recombination to form the pYPK0_PDC1_KlLAC4_PGI1 circular plasmid.

The same procedure was performed for the LAC12 but with the pYPKa_Z_PGI1 vector for the promoter and pYPKa_E_TPI1 for terminator. The resulting vector is called pYPK0_PGI1_KlLAC12_TPI1.

The two expression cassettes were pcr amplified and joined by homologous recombination with a pYPKpw vector digested with ZraI resulting in the pYPK0_PDC1_KlLAC4_PGI1_KlLAC12_TPI1 vector expressing both genes.

In this example, each file contain only one snippet, but all snippets could be put in one file.

The advantage of separate files is that they can be reused.

Each file name starts with the header of the snippet, like "pcr_...". This is optional, but makes it easier to sort the files.