EGB25 - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

original artwork

The objective of the lab course is to construct a new plasmid cloning vector called pTA7. This vector will be an E. coli / Saccharomyces cerevisiae shuttle vector. This vector will be useful for cloning using the Yeast Pathway Kit, see the next section "Background" "for more details.

In the mec research group, we are interested in understanding and engineering the biosynthesis of fatty acids and related products by the unicellular fungi known as baker's yeast S. cerevisiae.

Genetic engineering of complex traits require the simultaneous deletion and/or expression of multiple genes. This is a challenging problem as genetic engineering is time consuming. To solve this problem, we developed a protocol for the assembly of metabolic pathways that we call the Yeast Pathway Kit (YPK). See our publication in ACS Synthetic Biology for more details.

We use this protocol for quick construction and expression of large metabolic pathways in baker's yeast Saccharomyces cerevisiae such as this heterologous fatty acid synthesis pathway. Plasmids such as the one we will construct in this lab course are used to propagate these constructs in E. coli or S. cerevisiae.

In order to do this they need at least:

• a selection marker for E. coli
• a selection marker for S. cerevisiae.
• an origin of replication for E. coli
• an origin of replication for S. cerevisiae.

The first plasmid we used for this purpose in YPK was called pYPKpw and it has the following functional parts (Table#1):

The Δcrp is an E. coli gene which is inactive and only provide a recombination site. The pathways that we make are meant for S. cerevisiae, but we often need to transfer the pathway to E. coli so we can obtain larger amounts of higher quality DNA for analysis or transformation.

The pUC origin of replication (ORI) results in a high copy number of the vector in E. coli which is an advantage for obtaining large amounts of DNA. However, we have observed genetic instability in E. coli for some large pathways that we suspect is linked to high copy number. Our experience is that a lower copy number provides more stability.

We conceived a series of plasmid vectors called pTAx where x is a number from 1 to 11 (at the moment). The pTAx vectors were designed to have a lower copy number in E. coli to try to solve the stability problems of pYPKpw. The pTAx plasmids should have a lower copy number in E. coli than the pUC based pYPKpw since they have the pBR origin of replication that includes the ROP gene. The first pTAx plasmid, pTA1 was constructed by a former post-doc in the group, Tatiana Andrevna, hence the name.

The pTAx vectors are made from five genetic elements (Table #2), see (pTAx assembly strategy).

Each element is a distinct segment from a particular source plasmid. The pTAx vectors are similar to each other, but differ in the selection markers (yeast marker) and yeast origin of replication (yeast ORI).

The lab course is divided into nine practical classes. Each student attends three of the nine classes.

material#LAB1

Summary:

• Plasmid miniprep using alkaline lysis

We will prepare four plasmids using alkaline lysis mini prep (see Table#3). These plasmids are the source for each of the five genetic elements. One plasmid is used as the source of two elements. The last column states why we need each particular plasmid.

We will use a homemade alkaline lysis plasmid miniprep protocol. Here is a short protocol for printing.

The teacher has prepared E. coli cultures in liquid or on solid medium beforehand.

Cultures with each plasmid were grown in or on LB with antibiotics for selection of the plasmids.

These videos show how to prepare plasmid DNA from E. coli using basically the same protocol.

material#LAB2

Summary:

• Gel#1 on plasmid DNA.
• Plasmid DNA dilution.
• Preparation of a PCR reaction.
• Make liquid YPD medium for LAB3.
• Make solid SD medium for LAB4.

1. Unfreeze the plasmids from LAB1
2. Vortex the tube to mix the content. If necessary, spin the tube for ~2-3 seconds to collect the liquid at the bottom.
3. Take out 10 µL of the content to a fresh tube.
4. Add 2 µL 6 x loading buffer to your plasmid DNA.
5. Put the gel in the gel tray in a square petri dish. The gel should have at least one well per group number and one extra for the marker.
6. Add 5 µL of the plasmid DNA to an empty well, start with the leftmost well.
7. All group members should load their plasmid.
8. Take note of where your samples are.
9. The teacher will help you to put the gel in the electrophoresis chamber.
10. The teacher will load the molecular weight marker.
11. Apply the electrical field as soon as you are done.
12. The electrophoresis last around ⌛15 min at ⚡200 volts in the Bachman gel tank using a homemade rectifier.
13. When the gel run is completed, the teacher with take a picture using a transilluminator.

• Put the gel in TAE + Midori Green, incubate 15-30 min
• Take picture

1. Pipette 1 mL (1000 µL) ultra-pure water into a clean 1.5 mL Eppendorf tube
2. Mark this tube well so that you can identify it.
3. Transfer 5 µL of the plasmid DNA to the tube with 1 mL water This will make a 5 µL/1000 µL = x 200 dilution.
4. Vortex the tube with the x 200 plasmid dilution so that the content is well mixed.
5. Put the tube with concentrated plasmid DNA back into the freezer.
6. Leave the tube with your diluted plasmid DNA on the bench and continue with the next step.

Each student should prepare one PCR reaction. Add the reagents in the order given below in a 200 µL PCR tube. Pipette very carefully, there is only a limited amount of reagent which is expensive.

PCR amplification (50 µL):

• 33 µL 1.66 x PCR mastermix (= 2x PCR mastermix with 6x loading buffer , hhis is a green solution that has both PCR master mix and loading buffer)
• 5 µL primer 1 (5 µM, check the google sheet for your PCR reaction)
• 5 µL primer 2 "
• 7 µL of the x 200 diluted plasmid DNA.

See see pTAx assembly strategy

The teacher will take the tubes to the BioRad T100 thermal cycler in the LGM laboratory.

Each group should make 100 mL liquid YPD medium in a 250 mL Schott flask. Put a small piece of autoclave tape on the lid. Label the flask properly (Content, Date, Course, Turno, Group etc.).

material#LAB3

Summary:

• Analyze PCR products from LAB2 on gel#2
• Inoculate S. cerevisiae in liquid YPD cultures with from LAB2

It is critical that you keep track of your PCR tube. They are very small and have only a number written on them. We need them for the transformation later, so we can not lose them.

1. Put the gel in the gel tray in a square petri dish. The gel should have at least one well per group number and one extra for the marker.
2. Add 5 µL of the PCR product DNA to an empty well, start with the leftmost well.
3. All group members should load their PCR product.
4. Take note of where your PCR product is.
5. The teacher will help you to put the gel in the electrophoresis chamber.
6. The teacher will then load the molecular weight marker.
7. Apply the electrical field as soon as you are done.
8. The electrophoresis last around 15 min at 200 volts in the Bachman gel tank using a homemade rectifier.
9. When the gel run is completed, the teacher with take a picture using a transilluminator.

The first task is to measure the optical density of the culture. We do this by diluting the culture ten times in the same medium and measuring the OD640 nm against the medium as blank.

1. Add some YPD medium to a plastic weighing boat. This medium does not need to be sterile.
2. Add 900 µL YPD from the weighing boat to a cuvette.
3. Mix cell culture and add 100 µL to the cuvette.
4. Measure OD640 with a spectrophotometer.
5. Calculate the optical density of the culture.
6. Calculate the volume of cells from the pre-culture that needs to be added to 40 mL of YPD medium to attain an OD640 = 0.17 (OD640 = 0.17 = 5 x 106 cells/ml using a spectrophotometer GENESYS20.)

We can calculate the culture volume we need to add by the equation below. This is a simple mass balance:

$Va \cdot OD640 = 0.17 \cdot (40 + Va)$

• Va is the volume we use to inoculate (mL).
• OD640 is the optical density for the the culture from which we inoculate.
• The culture volume before inoculating is 40 mL.
• The final optical density we want is 0.17.

If we rearrange the equation: $Va = \frac{6.80}{OD640 - 0.17}$

1. Wipe down the bench with ethanol and light the lamp to create a sterile environment.
2. Add 40 mL of YPD medium to a 50 mL FALCON tube.
3. Add $Va$ mL of pre-culture to your tube.
4. Mix and measure the OD640 by removing one mL to an empty cuvette
5. If the OD640 is ok, pour all the contents of the tube into a sterile Erlenmeyer flask.
6. Incubate until cells have grown for two generations i.e. final OD640 should be 0.17 * 2 * 2 = 0.68
7. Pour the cells into a sterile 50 mL FALCON tube and store on ice.

Each group should make 500 mL solid SD medium in a 1 L Schott flask. Put a small piece of autoclave tape on the lid. Label the flask properly, (Content, Date, Course, Turno, Group etc.).

material#LAB4

Summary:

• Wash competent yeast cells
• Preparation of DNA mixture
• Yeast transformation
• Plate transformants on solid SD medium from LAB3

1. Decant the supernatant slowly without disturbing the cells.
2. Pour the content of the yeast culture into a 1.5 mL Eppendorf tube
3. Spin for 20 s in a micro-centrifuge at top speed
4. Remove supernatant by decanting (pipette not needed).
5. Add 1 mL ultra pure water.
6. Resuspend cells by pipetting slowly with a P1000 pipette (slowly, don't make froth).
7. Spin for 20 s in a micro-centrifuge at top speed
8. Remove supernatant by decanting (pipette not needed).
9. Add 800 µL ultra pure water.
10. Resuspend cells by pipetting slowly with a P1000 pipette (slowly!).
11. Put the tube on ice.

We need two mixtures, one complete that we call "➕" and one that lacks the pBR fragment that we call "🔺" (delta)", see Table #5. The ∆ mix is a negative control where we would expect few transformants. The plan is for 12 students to transform with the "➕" mix and 4 students with the "🔺" mix.

1. Pool all successful PCR products together except the tubes with the pBR fragment.
2. Measure the volume using a pipette ( for example 380 µL)
3. Divide the mixture 3/4 (➕) and 1/4 (🔺) (285 µL and 95 µL)
4. Add the pBR fragment to the ➕ mix (70 µL)
5. Add an equal portion of water to the 🔺 mix (70/3 = 23 µL)
6. Add 500 µL water to the ➕ mix (to have enough volume for 12 x 60 µL)
7. Add 167 µL water to the 🔺 mix (to have enough volume for 4 x 60 µL)
8. Mix by vortexing.
9. Divide ➕ mix into 3 tubes, 285 µL in each (one for each group).

Each student should make one transformation. This protocol is described in detail here.

1. Mix washed cells by inverting the tube.
2. Transfer 67 µL of the cell suspension to a clean 1.5 mL Eppendorf tube per group member (If the group has four members, you need four tubes.). Mark the tubes with your initials.
3. Centrifuge the cells for 20s at the highest speed of the microcentrifuge.
4. Remove supernatant with a P200 pipette. Leave the cell pellet at the bottom of the tube, do not resuspend.
5. Add 40 µL of ➕ or 🔺 DNA mix to the tube with cells.
6. Add 200 µL PLS (PEG-LiAc-ssDNA). Be careful and pipette slowly as PLS is sticky. Use a P1000 pipette with blue tip.
7. Vortex the tubes until cells are well resuspended and no clumps visible.
8. Put the tubes in a floating tube rack at 42°C.
9. Incubate for 40 min.
10. Mark a Petri dish with the appropriate solid medium with your group number and name. Write on the back side of th eplate, not on the lid.
11. Add about 1/2 mL glass spheres (~10-15 spheres) to the Petri dish.
12. Time for ☕ break.
13. Remove tube from water bath after 40 min and put tube on ice for at least 2 min.
14. Spin tube for 20s at highest speed.
15. Remove supernatant with a P200 pipette. Leave the cell pellet at the bottom of the tube.
16. Add 300 µL YPD medium and resuspend with the pipette by slowly pipetting up and down. Be careful as the cells are sensitive
17. Transfer 100 µL of the cell suspension to your Petri dish.
18. Spread the cells by shaking the glass spheres (The samba method).
19. Give the rest of the cell suspension to the instructor
20. Incubate the plates upside down for 2-4 days at 30°C.

material#LAB5

The objective of this task is to simulate the outcome of each of the five PCR product used to make the vector.

1. The primer numbers and template plasmids used can be found in Table#4 above.
2. The template sequences are available through links in Table 1.
3. The primer sequences for each primer number can be found in Table 3.
4. Simulate the PCR products using primer sequences and
5. Add the size and ldseguid checksum for each of the PCR products to the LAB5 tab in the course Google sheet.

1. Use the Assembly simulator (here) to join the five sequences.
2. Annotate your sequence with pLannotate
3. Add the size and cdseguid checksum for your plasmid as well as the sequence to the LAB5 tab in the Course Google Sheet.

Pick one isolated colony with a sterile pipette tip and inoculate a Petri dish with the the appropriate solid medium.

material#LAB6

Summary:

• NaOH total yeast DNA preparation
• Colony PCR

Each student should have a plate with colonies. Do not contaminate this plate, we will need these yeast cells later.

1. Watch this YouTube video (2 min) describing the technique.
2. Add 20 µL 20 mM NaOH to a clean 1.5 mL Eppendorf tube.
3. Pick a small amount of cells from your plate (from LAB5) with a yellow tip. It is important not to take too much and no agarose (see at 52 s in the video).
4. Add the cells to the solution and swirl to mix (see at 59 s in the video).
5. Incubate tubes at 95°C for ten minutes.
6. When the 95°C incubation is over, add 180 µL TE buffer.
7. Vortex the tube for 30 s.
8. Spin at max speed in a micro-centrifuge for 10-20 s.

1. Add 15 µL of PCR mix^* to a new PCR tube (these are the small tubes).
2. Add 5 µL of the yeast DNA to the PCR tube, do not disturb the cell debris from the bottom of the tube.
3. Put tubes in PCR machine (or freeze the tubes at -20°C for later).
4. Run this PCR program:

Taq DNA pol
|95°C  |95°C               |     |
|______|_____          72°C|72°C |
|10min |30s  \ 53.8°C _____|_____|
|      |      \______/ 0:30|5 min|
|      |          30s      |     |

^* PCR mix for 25 reactions (15 µL for a 20 µL PCR reaction): 5. 25 * 13 = 260 µL 1.5x Green PCR mastermix 6. 25 * 1 µL = 25 µL 1222 (10 µM) 7. 25 * 1 µL = 25 µL 1779 (10 µM)

Summary:

• Run gel with colony PCR products from LAB6
• Inoculate 1 mL yeast cultures for plasmid rescue from liquid medium.
• Prepare solid LB Lennox medium for LAB8

1. Put a piece of gel in a square Petri dish
2. Load 8 µL of the the PCR product.
3. Run the gel for 15 - 20 min in TAE buffer.

While the gel is running, each group should prepare 250 mL solid LB Lennox medium in a 500 mL Schott bottle.

See if any of the previously prepared liquid YPD medium (LAB2) is still fresh? Inoculate 1 mL YPD medium in a 2 mL Eppendorf tube from the yeast plate using a sterile pipette tip.

This culture is for plasmid rescue next week.

material#LAB8 Summary:

• Prepare crude yeast DNA from cultures prepared in LAB7.
• Transform E. coli with crude yeast DNA.
• Plate E. coli on Petri dishes with solid LB medium from LAB7

1. Each student has at least one Eppendorf tube with S. cerevisiae frozen cells from LAB7.
2. Add 200 µL P1 solution to the frozen cells
3. Add 200 µl of glass beads (about one full 0.2 mL PCR tube).
4. Resuspend cells by vortexing briefly.
5. Vortex the tubes for 5 minutes using a disruptor genie.
6. Add 200 µL of P2 solution (use googles, solution has NaOH). Do this as soon as possible, the disruption of the cells liberate nucleases that can damage DNA.
7. Invert tube slowly for 3 min.
8. Add 200 µL P3 solution.
9. Invert tube slowly for 3 min.
10. Spin (centrifuge) tubes for 10 min at highest speed.
11. While the tubes are spinning, prepare one 1.5 mL Eppendorf tube with 1 mL 96-100% ethanol
12. Transfer 400-500 µL of the supernatant from the centrifugation to the tube with ethanol.
13. Invert tube slowly ~10 times to mix.
14. Spin (centrifuge) tubes for 10 min at highest speed.
15. Remove all liquid by decanting.
16. Add 1 mL 70% ethanol to the tube
17. Spin (centrifuge) tubes for 1 min at highest speed.
18. Remove all liquid by decanting.
19. Remove as much liquid as possible with a pipette, spin again if necessary.
20. Dry the DNA for 5 min at 50°C.
21. Add 50 µL of TE buffer to the precipitated DNA and vortex briefly to dissolve.
22. Label your tube with the number in the Google sheet next to your name.

1. Add the 10 µL of the plasmid DNA to the tube with competent cells, flick the tube a few times to mix. Do NOT vortex the cells at this point.
2. prepare a cup with ice/water slurry.
3. Incubate for 5-10 min on ice.
4. Heat shock in water bath at 42°C during EXACTLY 45 s ⏱️.
5. Cool the tube for 2 min in a water/ice slurry for fast heat transfer.
6. Add 300 µL pre-warmed (37°C) liquid LB medium.
7. Add 20 µL ampicillin to the cells.
8. Mix cells by pipetting slowly up and down.
9. Plate 200 µL by adding 20 - 30 sterile glass beads to an LB plate and swirl to spread the cells.
10. Incubate inverted at 37°C for 18 - 24 hours. This protocol is described in greater detail here.

• Prepare crude yeast DNA from cells with glass beads and an E. coli plasmid miniprep kif
• Transform E. coli with crude yeast DNA
• Plate E. coli on Petri dishes with solid LB-amp medium from LAB7

1. (Scrape some yeast cells off a plate)
2. (Add 1 mL water in a 2 mL Eppendorf tube)
3. (Wash cells a and add glass beads)
4. (Add 200 µl of glass beads (about one full 0.2 mL PCR tube) to a tube)
5. (Add the amount of resuspension solution to the cells indicated by the kit)
6. Vortex the tubes for 5 minutes using a disruptor genie.
7. Add the lysis buffer as soon as possible, the disruption of the cells liberate nucleases that can damage the DNA.
8. Follow the rest of the alkaline lysis mini-prep kit protocol. .
9. If the kit has an optional wash step to get rid of nucleases, use that.
10. Elute in a small volume 50 µL or 30 µL for higher concentration.

1. Add the 10 µL of the plasmid DNA to the tube with competent cells, flick the tube a few times to mix. Do NOT vortex the cells at this point.
2. Incubate for up to 30 min on ice.
3. Heat shock in water bath at 42°C during EXACTLY 45s.
4. Cool the tube for 1-2 min in a water/ice slurry for fast heat transfer.
5. Add 1 mL pre-warmed liquid LB medium to one and proceed to the next step or let cells recover at 37°C for 1 h if time allows.
6. Pipette 300 µL of the content to a new Eppendorf tube. Give the remaining cells to the teacher.
7. Add 20 µL ampicillin (x1000) and mix by inversion
8. Pipette all of the content to a LB plate with 10-20 sterile glass beads and swirl the plate to spread the liquid.
9. Incubate plates inverted for 18-24 h at 37°C

material#LAB10

Summary:

• cut DNA with restriction enzymes, one enzyme per group.
• Plasmid miniprep using a commercial kit for DNA sequencing.
• Run agarose gel#5 on plasmid DNA and digested plasmid DNA

Enzyme mix = 5 µL Enzyme + 5 µL buffer x10

• Pipette 8 µL of the plasmid solution into a new Eppendorf tube.
• Mark the Eppendorf tube
• Ask your teacher to add 2 µL of Restriction enzyme mix (Table#6) to your tube.
• Spin and incubate for one hour at 37°C. During this incubation, do the plasmid preparation below.

Preparation of plasmids using a commercial alkaline lysis mini prep kit. The teacher has prepared E. coli cultures in liquid medium beforehand. Cultures with each plasmid were grown in or on LB with antibiotics for selection of the plasmids. We will use the NZYMiniprep.

• Put a 2 µL drop of 6x DNA loading buffer on a piece of Parafilm.
• Add 6 µL of plasmid DNA to the drop
• Put the gel in the gel tray.
• Add more buffer (if needed) until the gel is just submerged.
• Add all of the DNA to an empty well, start with the leftmost well.
• Take note of where your samples are.
• The teacher will load the molecular weight marker.
• Apply the electrical field as soon as you are done.
• The electrophoresis last around 15 - 20 min.
• When the gel run is completed, the teacher with take a picture using a transilluminator.

 [[EGB25_002-1.png]]

Gel: NZYMiniprep, elution 50 µL warm AE buffer.

LOCUS       pTA7                    5450 bp    DNA     circular UNK 01-JAN-1980
DEFINITION  amp pbr crp∆ 2µ TRP1.
ACCESSION   id_rc
VERSION     id_rc
KEYWORDS    .
SOURCE      
  ORGANISM  .
            .
COMMENT     pydna cdseguid=xbRMZpY-PTfFNKgRULL-_Na0Pu0 2024-06-07T15:21:53
            ZraI:1
            AatII:1
            EcoRV:2
            FspAI:1
FEATURES             Location/Qualifiers
     primer_bind     complement(66..94)
                     /label="865_pYPKpwR"
                     /ApEinfo_fwdcolor="#baffa3"
                     /ApEinfo_revcolor="#ffbaba"
     primer_bind     114..143
                     /label="866_pYPKpwF"
                     /ApEinfo_fwdcolor="#baffa3"
                     /ApEinfo_revcolor="#ffbaba"
     promoter        complement(262..281)
                     /note="pLannotate"
                     /database="snapgene"
                     /identity="100.0"
                     /match_length="44.4"
                     /fragment="True"
                     /other="promoter"
                     /locus_tag="T5 promoter (fragment)"
                     /label="T5 promoter (fragment)"
                     /ApEinfo_label="T5 promoter (fragment)"
                     /ApEinfo_fwdcolor="#EE92FE"
                     /ApEinfo_revcolor="#EE92FE"
                     /ApEinfo_graphicformat="arrow_data {{0 0.5 0 1 2 0 0 -1 0
                     -0.5} {} 0} width 5 offset 0"
     primer_bind     complement(339..355)
                     /label="977_Crp.REV"
                     /PCR_conditions="primer
                     sequence:TACAATAGAGTTCCGAGGTAAACGCTTTTCGTTCTTGTCTCATTGCC"
                     /ApEinfo_fwdcolor="#baffa3"
                     /ApEinfo_revcolor="#ffbaba"
     misc_binding    386..389
                     /bound_moiety="echinomycin"
     misc_binding    401..404
                     /bound_moiety="echinomycin"
     misc_binding    412..415
                     /bound_moiety="echinomycin"
     misc_binding    417..420
                     /bound_moiety="echinomycin"
     misc_binding    429..432
                     /bound_moiety="echinomycin"
     primer_bind     446..470
                     /label="1113_Amp.fw.nw"
                     /PCR_conditions="primer
                     sequence:GAAAAGCGTTTACCTCGGAACTCTATTGTAGAACCCCTATTTGTTTATTT
                     TTCTA"
                     /ApEinfo_fwdcolor="#baffa3"
                     /ApEinfo_revcolor="#ffbaba"
     regulatory      506..512
                     /regulatory_class="promoter"
                     /note="promoter P3 (6)"
     regulatory      535..539
                     /regulatory_class="ribosome_binding_site"
                     /note="Shine-Dalgarno sequence"
     gene            547..1407
                     /gene="bla"
     CDS             547..1407
                     /gene="bla"
                     /note="E-286"
                     /codon_start=1
                     /transl_table=11
                     /product="beta-lactamase"
                     /protein_id="AAB59737.1"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     sig_peptide     547..615
                     /gene="bla"
     mat_peptide     616..1404
                     /gene="bla"
                     /product="beta-lactamase"
     primer_bind     complement(1436..1457)
                     /label="987_Amp.REV"
                     /PCR_conditions="primer
                     sequence:AGAAAGTCTACACCTTACGCTGATTGGATTTGAAGTTTTAAATCAATCTA
                     AA"
                     /ApEinfo_fwdcolor="#baffa3"
                     /ApEinfo_revcolor="#ffbaba"
     primer_bind     1487..1505
                     /label="1196_Pbr.FW"
                     /PCR_conditions="primer
                     sequence:AATCCAATCAGCGTAAGGTGTAGACTTTCTCTGTCAGACCAAGTTTACT"
                     /ApEinfo_fwdcolor="#baffa3"
                     /ApEinfo_revcolor="#ffbaba"
     repeat_region   complement(1594..1631)
                     /note="corresponds to one of the 38bp repeats found in Tn3
                     (bp 1-38 and complement (4920-4957))"
                     /rpt_type=inverted
     old_sequence    complement(2049..2050)
                     /note="revision according to (17)"
                     /citation=[2]
                     /citation=[17]
                     /replace="at"
     old_sequence    complement(2049)
                     /note="revision according to (16)"
                     /citation=[2]
                     /citation=[16]
                     /citation=[17]
                     /replace="t"
     old_sequence    complement(2050)
                     /citation=[17]
     rep_origin      complement(2244)
     misc_binding    complement(2332..2340)
                     /bound_moiety="dnaA"
     misc_feature    2365..2428
                     /note="L-strand Y effector site"
                     /citation=[5]
     repeat_region   2530..2534
                     /note="gamma-delta insertion target sequence"
                     /rpt_type=direct
     misc_feature    complement(2612..2768)
                     /note="H-strand Y effector site"
                     /citation=[5]
     CDS             complement(2673..2864)
                     /codon_start=1
                     /transl_table=11
                     /product="ROP protein"
                     /protein_id="AAB59736.1"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     old_sequence    complement(2864..2865)
                     /citation=[17]
     misc_difference complement(2865..2866)
                     /note="conflict"
                     /citation=[23]
                     /replace="caa"
     regulatory      complement(2869..2874)
                     /regulatory_class="ribosome_binding_site"
     regulatory      complement(2870..2874)
                     /regulatory_class="ribosome_binding_site"
                     /note="Shine-Dalgarno sequence"
     old_sequence    complement(2886..2887)
                     /citation=[2]
                     /citation=[22]
     misc_difference complement(2887..2888)
                     /note="conflict"
                     /citation=[23]
                     /replace="att"
     primer_bind     2895..2913
                     /label="984_Micron.FW"
                     /PCR_conditions="primer
                     sequence:ATCGTATCTGCTGCGTAAATAGTAGTCAACGATCGTACTTGTTACCCAT"
                     /ApEinfo_fwdcolor="#baffa3"
                     /ApEinfo_revcolor="#ffbaba"
     primer_bind     complement(2910..2924)
                     /label="1195_Pbr.REV"
                     /PCR_conditions="primer
                     sequence:GTTGACTACTATTTACGCAGCAGATACGATCTCGTTTCATCGGT"
                     /ApEinfo_fwdcolor="#baffa3"
                     /ApEinfo_revcolor="#ffbaba"
     rep_origin      3134..4480
                     /note="yeast 2μ plasmid origin of replication"
                     /note="color: #ffff00"
                     /label="2μ ori"
     primer_bind     complement(4461..4478)
                     /label="983_Micron.REV"
                     /PCR_conditions="primer
                     sequence:CAGAGCAGACAGTTCCTTTACGAGATTTTAGATCCAATATCAAAGGAA"
                     /ApEinfo_fwdcolor="#baffa3"
                     /ApEinfo_revcolor="#ffbaba"
     primer_bind     4569..4589
                     /label="1804_TRP1fp_pTA"
                     /PCR_conditions="primer
                     sequence:TAAAATCTCGTAAAGGAACTGTCTGCTCTGtataaaaataggcgtatcac
                     g"
                     /ApEinfo_fwdcolor="#baffa3"
                     /ApEinfo_revcolor="#ffbaba"
     promoter        4595..4696
                     /gene="S. cerevisiae TRP1"
                     /label="TRP1 promoter"
                     /note="color: #ffffff"
     CDS             4697..5371
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /note="yeast auxotrophic marker"
                     /note="color: #ff7f50"
                     /product="phosphoribosylanthranilate isomerase, required
                     for tryptophan biosynthesis"
                     /transl_table=1
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK*"
                     /label="TRP1"
     rep_origin      complement(5210..5450)
                     /note="S. cerevisiae autonomously replicating sequence
                     ARS1/ARS416"
                     /note="color: #ffff00"
                     /label="ARS1"
     primer_bind     5421..5436
                     /label="978_Crp.FW"
                     /PCR_conditions="primer
                     sequence:AACTGTAAAATCAGGTATCTCGTAGTCCGTGTTCTGATCCTCGAGC"
                     /ApEinfo_fwdcolor="#baffa3"
                     /ApEinfo_revcolor="#ffbaba"
     primer_bind     complement(5430..5450)
                     /label="1347_TRP1rp_pTA"
                     /PCR_conditions="primer
                     sequence:ACGGACTACGAGATACCTGATTTTACAGTTGATCTTTTATGCTTGCTTTT
                     C"
                     /ApEinfo_fwdcolor="#baffa3"
                     /ApEinfo_revcolor="#ffbaba"
ORIGIN
        1 gttctgatcc tcgagcatct taagaattcg tcccacggtt tgtctagagc agccgacaat
       61 ctggccaatt tcctgacggg taattttgat ttgcatgccg tccgggtgag tcatagcgtc
      121 tggtgacgtc atgcgcatga tatcttcaca ggcggttttc gcacgtaccc atgcgctacg
      181 ttcctggccc tcttcaaaca ggcccagttc gccaataaaa tcaccctgat tcagatagga
      241 gaggatcatt tctttaccct cttcgtcttt gatcagcact gccacagagc ctttaacgat
      301 gtagtacagc gtttccgctt tttcaccctg gtgaataagc gtgctcttgg atgggtactt
      361 atgaatgtgg caatgagaca agaacgaaaa gcgtttacct cggaactcta ttgtagaacc
      421 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
      481 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
      541 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
      601 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
      661 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
      721 acttttaaag ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa
      781 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
      841 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
      901 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
      961 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     1021 gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg
     1081 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     1141 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     1201 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     1261 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     1321 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     1381 tcagaccaag tttactcata tatactttag attgatttaa aacttcaaat ccaatcagcg
     1441 taaggtgtag actttctctg tcagaccaag tttactcata tatactttag attgatttaa
     1501 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     1561 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     1621 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     1681 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     1741 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     1801 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     1861 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     1921 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     1981 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     2041 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     2101 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2161 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2221 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     2281 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     2341 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     2401 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca
     2461 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     2521 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     2581 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     2641 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     2701 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     2761 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     2821 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     2881 accgatgaaa cgagatcgta tctgctgcgt aaatagtagt caacgatcgt acttgttacc
     2941 catcattgaa ttttgaacat ccgaacctgg gagttttccc tgaaacagat agtatatttg
     3001 aacctgtata ataatatata gtctagcgct ttacggaaga caatgtatgt atttcggttc
     3061 ctggagaaac tattgcatct attgcatagg taatcttgca cgtcgcatcc ccggttcatt
     3121 ttctgcgttt ccatcttgca cttcaatagc atatctttgt taacgaagca tctgtgcttc
     3181 attttgtaga acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag aatctgagct
     3241 gcatttttac agaacagaaa tgcaacgcga aagcgctatt ttaccaacga agaatctgtg
     3301 cttcattttt gtaaaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct
     3361 gagctgcatt tttacagaac agaaatgcaa cgcgagagcg ctattttacc aacaaagaat
     3421 ctatacttct tttttgttct acaaaaatgc atcccgagag cgctattttt ctaacaaagc
     3481 atcttagatt actttttttc tcctttgtgc gctctataat gcagtctctt gataactttt
     3541 tgcactgtag gtccgttaag gttagaagaa ggctactttg gtgtctattt tctcttccat
     3601 aaaaaaagcc tgactccact tcccgcgttt actgattact agcgaagctg cgggtgcatt
     3661 ttttcaagat aaaggcatcc ccgattatat tctataccga tgtggattgc gcatactttg
     3721 tgaacagaaa gtgatagcgt tgatgattct tcattggtca gaaaattatg aacggtttct
     3781 tctattttgt ctctatatac tacgtatagg aaatgtttac attttcgtat tgttttcgat
     3841 tcactctatg aatagttctt actacaattt ttttgtctaa agagtaatac tagagataaa
     3901 cataaaaaat gtagaggtcg agtttagatg caagttcaag gagcgaaagg tggatgggta
     3961 ggttatatag ggatatagca cagagatata tagcaaagag atacttttga gcaatgtttg
     4021 tggaagcggt attcgcaata ttttagtagc tcgttacagt ccggtgcgtt tttggttttt
     4081 tgaaagtgcg tcttcagagc gcttttggtt ttcaaaagcg ctctgaagtt cctatacttt
     4141 ctagctagag aataggaact tcggaatagg aacttcaaag cgtttccgaa aacgagcgct
     4201 tccgaaaatg caacgcgagc tgcgcacata cagctcactg ttcacgtcgc acctatatct
     4261 gcgtgttgcc tgtatatata tatacatgag aagaacggca tagtgcgtgt ttatgcttaa
     4321 atgcgtactt atatgcgtct atttatgtag gatgaaaggt agtctagtac ctcctgtgat
     4381 attatcccat tccatgcggg gtatcgtatg cttccttcag cactaccctt tagctgttct
     4441 atatgctgcc actcctcaat tggattagtc tcatccttca atgctatcat ttcctttgat
     4501 attggatcta aaatctcgta aaggaactgt ctgctctgta taaaaatagg cgtatcacga
     4561 ggccaattcg gtcgaaaaaa gaaaaggaga gggccaagag ggagggcatt ggtgactatt
     4621 gagcacgtga gtatacgtga ttaagcacac aaaggcagct tggagtatgt ctgttattaa
     4681 tttcacaggt agttctggtc cattggtgaa agtttgcggc ttgcagagca cagaggccgc
     4741 agaatgtgca ctagattccg atgctgactt gctgggtatt atatgtgtgc ccaatagaaa
     4801 gagaacaatt gacccggtta ttgcaaggaa aatttcaagt cttgtaaaag catataaaaa
     4861 tagttcaggc actccgaaat acttggttgg cgtgtttcgt aatcaaccta aggaggatgt
     4921 tttggctctg gtcaatgatt acggcattga tatcgtccaa ctgcatggag atgagtcgtg
     4981 gcaagaatac caagagttcc tcggtttgcc agttattaaa agactcgtat ttccaaaaga
     5041 ctgcaacata ctactcagtg cagcttcaca gaaacctcat tcgtttattc ccttgtttga
     5101 ttcagaagca ggtgggacag gtgaactttt ggattggaac tcgatttctg actgggttgg
     5161 aaggcaagag agccccgaga gcttacattt tatgttagct ggtggactga cgccagaaaa
     5221 tgttggtgat gcgcttagat taaatggcgt tattggtgtt gatgtaagcg gaggtgtgga
     5281 gacaaatggt gtaaaagact ctaacaaaat agcaaatttc gtcaaaaatg ctaagaaata
     5341 ggttattact gagtagtatt tatttaagta ttgtttgtgc acttgcctgc aagccttttg
     5401 aaaagcaagc ataaaagatc aactgtaaaa tcaggtatct cgtagtccgt
//

TAAAATCTCGTAAAGGAACTGTCTGCTCTG s3



TAAAATCTCGTAAAGGAACTGTCTGCTCTGtataaaaataggcgtatcacg 1804_TRP1fp_pTA replaces 1348 & 1680
                              --------------------- YEplac112 YCplac22 YIplac204


               GAACTGTCTGCTCTGttcaagaattaattcggtcg  1680_TRP1fp_pTA New TRP1 primer, partial s3, replaces 1348, replaced by 1804
                                        ----------  YXplacs snapgene


TAAAATCTCGTAAAGGAACTGTCTGCTCTGtttgccgattaagaattcg   1348_TRP1fp_pTA  This primer was replaced by 1680
                              -------------------   TRP1 SGD
                                           ------   YXplacs snapgene


tataaaaataggcgtatcacgaggccctttcgtcttcaagaattaattcggtcgaaaaaagaaaaggagagggccaagagggagggcattggtgactattgagcacgtgagtatacgtgattaagcacacaaaggcagcttggagtatg TRP1 YEplac112 YCplac22
--------------------- 1804        -------------------- 1680
                                            ------     1348

tataaaaataggcgtatcacgaggcc==================aattcggtcgaaaaaagaaaaggagagggccaagagggagggcattggtgactattgagcacgtgagtatacgtgattaagcacacaaaggcagcttggagtatg TRP1 YIplac204
--------------------- 1804                  ---------- 1680
                                            ------     1348

                               TTTGCCGATTAAGaattcggtcgaaaaaagaaaaggagagggccaagagggagggcattggtgactattgagcacgtgagtatacgtgattaagcacacaaaggcagcttggagtATG TRP1 SGD
                               ------------------- 1348

The YEplac112, YCplac22 and YIplac204 vectors all express the TRP1 gene. The sequences for these vectors are available from both Genbank and Snapgene (see pTAx assembly strategy).

A peculiarity is that the YIplac204 TRP1 promoter seems to have an 18 bp deletion (marked by ==================) compared to the sequences in YEplac112 YCplac22.