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| ==Design== | | ==Design== |
| | The F plasmid origin needs to be designed. The complete F plasmid with partitioning genes in ~10kb in length. It contains several BioBricks restriction sites in both coding and noncoding regions. |
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| 5' -- VF2 -- 40 bases spacer sequence? -- plasmid barcode -- TOPO site -- BBa_B0055 -- BB prefix -- | | Once designed, the F plasmid origin can be assembled with an antibiotic resistance marker and cloned into the [[Synthetic Biology:Vectors/pSB**5 design|vector scaffold]] to generate a new single copy BioBricks plasmid. |
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| BBa_P1010 -- BBa_I50020 --
| | Chris Anderson suggested inclusion of the R6K origin in these plasmids (rather than inclusion of a pUC19 origin in the multiple cloning site). The R6K origin is a conditional origin. It only works in the presence of the trans-acting protein Π (encoded by pir) for replication. R6K replicates at a medium copy (15 per cell) in pir+ strains and high copy (250 per cell) in pir-116 (high-copy-number mutant) E. coli hosts. |
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| BBsuffix -- TOPO site -- BBa_B0054 -- VR -- BBa_I50000 -- antibiotic resistance cassette -- 3'
| | ==Fabrication== |
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| ===Proposed features===
| | *'''[[User:Rshetty|Reshma]] 15:27, 27 February 2007 (EST)''': Tom suggested that mutations of the BioBricks sites could be done via Pete Carr, Farren Isaacs and George Church's single stranded mutation method. |
| *F plasmid backbone | |
| *positive selection marker (i.e. ''ccdB'' or ''sacB'') in between BioBricks restrictions sites to facilitate cloning
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| *a high copy origin in the multiple cloning site to enable easy purification of the vector
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| *strong terminators flanking the BioBricks insertion site
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| *no loxP or cos insertion sites or Tn7 attachment sites?
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| **I can't think of an obvious use of these sites unless we want to build in the capability for integrating onto the genome. However, wouldn't it make more sense to build in recombination capabilities onto a higher copy number vector than this?
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| *no blue-white screening?
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| **inclusion of a ''lacZα'' fragment would restrict its use as a part
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| *multiple versions with different antibiotic resistance markers
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| *no selection system for mammalian cells
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| *VF2 and VR sites
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| *Unique tag near but outside the cloning sites for identification during sequencing. (from Randy)
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| *orient the antibiotic resistance cassette on the reverse strand from the BioBricks insertion site
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| *topoisomerase mediated TA cloning capability
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| | ==Notes== |
| ===Drawbacks=== | | ===Drawbacks=== |
| *Can only be used in F<sup>-</sup> strains | | *Can only be used in F<sup>-</sup> strains |
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| *It is unclear whether this vector would truly be operating at single copy. If it is not, perhaps it is easier to stick with the pSB2* plasmids. | | *It is unclear whether this vector would truly be operating at single copy. If it is not, perhaps it is easier to stick with the pSB2* plasmids. |
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| ==Planning== | | ==Relevant pages== |
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| ===Current status===
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| The following parts have been designed
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| *[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6307 BBa_I50000]: F plasmid backbone with BioBricks restriction sites removed
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| *[http://parts.mit.edu/r/parts/partsdb/view.cgi?part_id=6400 BBa_I50020]: high copy origin from pSB1A3
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| *[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6306 BBa_B0055]: upstream flanking terminator
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| *[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6305 BBa_B0054]: downstream flanking terminator
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| The following parts have been designed, fabricated and tested
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| *[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6370 BBa_P1001]: cassette providing tetracycline resistance (from [[Austin Che]])
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| *[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=6369 BBa_P1000]: cassette providing chloramphenicol resistance (from [[Austin Che]])
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| *[http://parts.mit.edu/r/parts/partsdb/view.cgi?part_id=4941 BBa_I1000] or [http://parts.mit.edu/r/parts/partsdb/view.cgi?part_id=6396 BBa_P1010]: ccd operon in BioBricks format (from Leon Chan)
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| ===To do list===
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| *One of the things needed for this project is BioBricked antibiotic resistance cassettes. This is also a prerequisite for the [[Standard E. coli Strain for BioBricks|standard strain project]]. Tom has ordered primers and is planning on cloning several resistance cassettes.
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| **I have TetR and CmR BioBricked using Tom's primers. --[[User:Austin|Austin]] 18:26, 3 Dec 2005 (EST)
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| *Design unique identifiers for vectors.
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| *Engineer in topoisomerase mediated TA cloning capability into the vector
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| ===To be decided===
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| *Choose between manual assembly of vector modules or direct synthesis of all plasmid variants
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| **Can we get a price break for synthesizing multiple plasmid variants?
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| **How many assemblies would we need to do?
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| **Is there a hybrid approach? Could we PCR the F plasmid backbone since its long and then have the collection of smaller parts (that would otherwise involve several assemblies) synthesized? Maybe a partial synthesis approach would help get around the issue of constructing a BioBricks insertion site?--[[User:Bcanton|BC]]
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| ***PCR'ing the F plasmid backbone is not very practical since there are several BioBricks sites in the backbone each of which would need to be individually mutated out. It is unlikely that there is anyone who is willing to do this much work. Therefore, the current plan is to synthesize the backbone. --[[Reshma Shetty | RS]]
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| *If all the vector components are specified in BioBricks format, how do we construct a BioBricks insertion site?
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| **Blunt-end ligation?
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| **Other restriction enzyme sites?
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| **PCR
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| **Use special restriction sites for vector construction (Austin's idea). Expanding on this, we could define a new idempotent assembly standard for exclusive use for vector components.
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| ===To be determined===
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| *Are we sure that F plasmids are really at 1-2 copies per cell? Why was pSB2K3-1 measured to be higher than that?
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| **From Johann Paulsson: it is unclear how tight of control F plasmid based vectors have over copy number fluctuation. Having the vector exist at single copy strongly depends on generation time. Faster growing cells are more likely to have multiple overlapping rounds of replications occurring simultaneously.
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| *What parts of the F plasmid are responsible for integration onto the genome? Can we omit them?
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| **cos and/or loxP sites are generally used for integration in the genome. Currently, I have no plans to include them in this vector.
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| *Many of the existing BACs only seem to have a partial ''sopC'' CDS, do we want the rest?
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| **pSMART VC vector appears to have a more complete ''sopC'' region. This may lead to tighter control of copy number.
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| *A set of orthogonal single copy replication origins to allow multiple vectors to be maintained in a cell. Can we have a set of vectors with F and P1 origins?--[[User:Bcanton|BC]] 17:36, 31 Oct 2005 (EST)
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| **Not sure this is possible. I believe the P1 origins use the par set of genes to maintain single copy whereas the F origins use the sop set of genes. The two sets are pretty homologous to eachother and therefore likely incompatible. I need to check on this more. --[[Reshma Shetty | RS]]
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| ** Perhaps derivatives from the two plasmids the Berkeley iGEM team used might permit two single copy vectors to be used simultaneously. --[[Reshma Shetty | RS]]
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| * Should the flanking terminators be placed outside the VF2 and VR primer binding sites? Is it useful to have them within? Moving the flanking terminators outside the primer binding sites means fewer bases to sequence through before hitting the part. Alternatively, we could move the terminators to just inside the primers since anyway ~40bp are needed before sequence data is of high quality.
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| ===Topoisomerase mediated TA cloning capability===
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| ====Topoisomerase site====
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| Topoisomerase recognition site
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| ---------
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| Topoisomerase nick site
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| V
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| C C C T T N N N N N N
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| G G G A A N N N N N N
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| ^
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| Restriction enzyme must nick here
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| ====Possible enzymes to generate 3' T overhang====
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| Each can accommodate the topoisomerase site CCCTT
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| *[http://www.neb.com/nebecomm/products/productR0600.asp BmrI]
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| **offset cutter that leaves 3' single base overhang
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| **two sites in sopC (incD) repeat region of BBa_I50000
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| **no sites in BBa_I50020
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| **no sites in BBa_P1010, BBa_P1000, BBa_B0055, BBa_B0054
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| **pretty high activity in all 4 NEB buffers
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| *[http://www.neb.com/nebecomm/products/productR0596.asp BciVI]
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| **offset cutter that leaves 3' single base overhang
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| **no sites in BBa_I50000
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| **one site in BBa_I50020
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| **no sites in BBa_P1010, BBa_P1000, BBa_B0055, BBa_B0054
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| *[http://www.neb.com/nebecomm/products/productR0533.asp XcmI]
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| **long recognition sequence with internal N<sub>9</sub> that leaves 3' single base overhang
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| **no sites in BBa_I50000
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| **no sites in BBa_I50020
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| **no sites in BBa_P1010, BBa_P1000, BBa_B0055, BBa_B0054
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| **100% activity only in NEBBuffer 2
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| Topoisomerase sites internal to vector components are not important because topoisomerase only operates near double stranded breaks.
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| Based on the preexistence of sites in the designed parts, XcmI looks like a good bet but it is not as robust to buffer conditions as BmrI.
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| ====Topoisomerase I====
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| Most of the papers reference Topoisomerase I from vaccinia. It seems to be available commercially at Epicentre. [http://www.epibio.com/item.asp?ID=274&CatID=112 html] [http://www.epibio.com/pdftechlit/108pl092.pdf protocol]
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| ====Vector preparation====
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| '''Reference'''<br>
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| J. A. Heyman, J. Cornthwaite, L. Foncerrada, J. R. Gilmore, E. Gontang, K. J. Hartman, C. L. Hernandez, R. Hood, H. M. Hull, W. Y. Lee, R. Marcil, E. J. Marsh, K. M. Mudd, M. J. Patino, T. J. Purcell, J. J. Rowland, M. L. Sindici, and J. P. Hoeffler. Genome-scale cloning and expression of individual open reading frames using topoisomerase i-mediated ligation. Genome Res, 9(1088-9051 (Print)):383–92, 1999. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10207160 pubmed]
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| *Vectors pcDNA3.1/GS and pYES2/GS
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| v
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| HindIII site
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| -----------
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| 5' N N N N N A A G C T T N N N N N 3'
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| 3' N N N N N T T C G A A N N N N N 5'
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| -----------
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| HindIII site
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| ^
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| *Cut with HindIII
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| 5' C C C T T A
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| 3' G G G A A T T C G A
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| A G C T T A A G G G 3'
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| A T T C C C 5'
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| *Ligate on oligos (TOPO-H and TOPO-4)
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| **By ligate, I think the paper really means anneal ... i.e. the backbone bond is not restored.
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| **TOPO-H destroys the HindIII site
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| **TOPO-4 provides recessed end
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| **In the drawings below, " | " refers to a break in the backbone on that strand.
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| TOPO-H oligo
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| -----------------------------------------------
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| 5' N N N N N A | A G C T C G C C C T T A T T C C G A T A G T G
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| 3' N N N N N T T C G A | G C G G G A
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| ------- -----------
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| HindIII overhang TOPO-4 oligo
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| TOPO-4 oligo HindIII overhang
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| ------------ -------
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| A G G G C G | A G C T T N N N N N 3'
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| G T G A T A C C T T A T T C C C G C T C G A A | N N N N N 5'
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| ------------------------------------------------
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| TOPO-H oligo
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| *Purify and cut again with HindIII to remove circular vector.
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| *Add TOPO5 oligo and topoisomerase.
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| **''Vaccinia'' topoisomerase I cleaves after and remains covalently attached to second T in CCCTT sequence.
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| Topoisomerase nick
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| v
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| TOPO-H oligo
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| -------------------------------------------------
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| 5' N N N N N A | A G C T C G C C C T T A T T C C G A T A G T G 3'
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| 3' N N N N N T T C G A | G C G G G A | A T A A G G C T A T C A C A A C 5'
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| ------- ----------- -------------------------------
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| HindIII overhang TOPO-4 oligo TOPO-5 oligo
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| TOPO-5 oligo TOPO-4 oligo HindIII overhang
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| ------------------------------- ------------ -------
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| C A A C A C T A T C G G A A T A | A G G G C G | A G C T T N N N N N 3'
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| G T G A T A C C T T A T T C C C G C T C G A | A N N N N N 5'
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| ------------------------------------------------
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| TOPO-H oligo
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| ^
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| Topoisomerase nick
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| *Add TOPO-10X stop buffer
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| *Purify away free oligonucleotides and unbound topoisomerase I
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| ==Notes==
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| See the [[Synthetic Biology:Vectors/Parts | list of parts for plasmid engineering]]. | | See the [[Synthetic Biology:Vectors/Parts | list of parts for plasmid engineering]]. |
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| [[Category:Project]] | | [[Category:Project]] |
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| | ==References== |
| | <biblio> |
| | #Jones-BiotechnolBioeng-1998 pmid=10099385 |
| | #Metcalf-Gene-1994 pmid=8125283 |
| | </biblio> |
