TITLE: nanochromosome arrays combinatory assembly
AUTHORS: Dariusz Abramczyk, Dariusz Abramczyk

[DESCRIPTION]
The yeast Pichia pastoris is used widely to biomanufacture high-value recombinant proteins. Its cells can secrete copious quantities of post-translationally modified proteins. Currently, however, heterologous genes must first be integrated into the genome in order to achieve expression.
We produced a synthetic 'chromosome-like' construct called here nanochromosome, an autonomously replicating and mitotically stable synthetic construct expressing heterologous genes. The nanochromosome contains essential scaffolding features as telomeres, centromere and yeast replication origins along with a versatile platform for genetic engineering. This platform could be used for the accurate and controlled insertion of multiple expression cassettes placed on 'landing pad',  in which an array of genes of interest alternate with ~1kp non-coding DNA sequences (LHR) chosen to facilitate simultaneous  double cross-overhomologous recombination and serve as spacers. The landing zone translates along the nanochromosome in an inchworming mode of sequential gene integrations that recycles a pair of antibiotic-resistance markers
 
  
  
Module with expression cassettes and LHR spacers as a pre-prepared in-vitro assembled DNA parts (insertion or integration arrays) is delivered to Pichia by transformation
Both the “integration” (receptor) and “insertion” (donor) arrays referred to herein consist of long, HR-ready, regions (LHRs) alternating with gene-expression cassettes. We constructed array parts containing either two or three LHRs e.g.LHRn-GoIn-AMRH/Z-LHRZ or LHRn-GoIn-LHRm-AMRH/Z-LHRZ, respectively, wherein: LHRn, LHRm and LHRZ are unique sequences of ~1000 bp from a library of LHRsA-Z, with LHRZ reserved as the last LHR in the array; GoIn  is a cassette consisting of a gene-of-interest with promoter and terminator regions; and AMRZ/H is a zeocin-resistance gene or hygromycin-resistance gene also with promoter and terminator regions. 
Preparation of ZeoR-LHRZ and HygR-LHRZ pair tandems in separate protocol https://www.protocols.io/view/preparation-of-parts-hygr-lhrz-and-zeor-lhrz-cqujvwun

The protocol for multi-DNA parts arrays preparation is based on combinatory assembly and molecular biology techniques customised to create a DNA parts (arrays) library in pUC19 plasmid. 

Major 3 experimental steps:

Step 1 - DNA parts generated by PCR with a complementary overhangs designed to create a combined linear dsDNA suitable for insertion into BsmBI-sites of pUC19 (DNA arrays library)
- a part generating by PCR, oligos deliver a complementary overhangs for digestion with BsmBI (Esp3I) or BsaI, 
- PCR product digestion with BsmBI or BsaI
- checking digested PCR products on agarose gel
- PCR clean up OR gel extraction of DNA and clean up (only in the case if a PCR product is not homogenous 
- estimation of DNA concentration (molar)
Note: (*) stands for AsiSI-FspI RE recognition site provides in Fd-oligo1 and RV-olig4

    
Step 2 - preparation of semi-products before the the final ligation with linearlized pUC adaptor plasmid
-  ligation part1+2 with T7 DNA ligase
- ligation part 3+4 with T7 DNA ligase
- loading ligation mixtures and separation on agarose gel, 
- gel extraction of estimated DNA band
-  agarose gel verification, DNA concentration (molar)

  
Step3 - the final ligation of pre-assembled parts with BsmBI-linearized pUC19
- part1+2 and part 3+4 and linearized pUC19 ligation (DNA T7 ligase)
- transformation into E.coli and selection on LB ampicillin plates
- verification by bacterial colony PCR 
- plasmid isolation and Sanger (Plasmidsaurus) sequencing
- positively verified clone storage in glycerol stock 

 
The assembled part can be retained by:
- PCR amplification using flanking oligonucleotides
- Digested by AsiSI or FspI (need to be checked for the absence RE site in internal array) and digestion mixture can be used for Pichia transformation

Note: LHR part (e.g. LHRA) in flanking region (as a part 1) sometimes called 'OUT' while the LHRA in internal site (as a part 3) sometimes called 'IN'


[STEPS]
SECTION: Preparation of triple-LHR integration array LHRA-PAOXPDI-LHRE-HygR-LHRZ
1. DNA parts preparation by PCR reactions
List of DNA parts 
 
* see 1.1 for prepartion (below)
**link https://www.protocols.io/view/preparation-of-parts-hygr-lhrz-and-zeor-lhrz-cqujvwun




SECTION: Preparation of triple-LHR integration array LHRA-PAOXPDI-LHRE-HygR-LHRZ
1.2. STEP 1 - PCR amplification of parts and digestion with suitable IIS RE

Parts amplified by PCR (using ProFlex PCR system, Applied Biosystems). Thermal Cycler

  
  
volumes in microliter
 
see oligo tables step 1 (above)

 5xQ5 buffer10101010betaine 5x1010101010mM dNTP (TAKARA)4444335/3382.5/2.5304/3052.5/2.5308/3182.5/2.5302/3032.5/2.5eDA199 100ng/ul1eDA125 100ng/ul1eDA176 100ng/ul1eDA89 100ng/ul1Q5 HF enzyme0.70.70.70.7water up to 50
 
 program Q5    Td-initial98oC30secTd98oC10sec33 cyclesTa58oC20secTe72oC6 min   Final extension72oC1 minhold4oChold
  
All PCR products purified by   and analysed on 1% agarose gel (with ) 

    
 
              size (bp)  concentration (ng/ul)  Molar concentration (nM)    LHRA  883  28  48    Poax1PDI  2905  64  34    LHRE  892  82  142    HygR-LHRZ  2583  101  60  
 

 Digestion with    or   

 
 1 2   33445 (test enzyme)6 (test enzyme)PCR  E 335/33846PCR D 304/30546PCR zeoC 308/318 (1)46PCR zeoC 308/318 (2)46PCR GFPcFH  302/303 (1)46PCR GFPcFH  302/303 (2)46puc19 nc22BsaI HF (20U/ul)20.5Esp3I (10U/ul))333330.5buf10x cutsmart66666666water 
 All PCR products purified by   and DNA concentration checked on DeNovix DS-11

DNA molar concentration calculation before ligation
 
            size (bp)  concentration (ng/ul)  Molar concentration (nM)    LHRA  883  40  70    Poax1PDI  2905  64  34    LHRE  892  82  142    HygR-LHRZ  2583  101  60  
 
SECTION: Preparation of triple-LHR integration array LHRA-PAOXPDI-LHRE-HygR-LHRZ
1.1. Prestep - Generation of pPICZA with POAX1PDIhis - (eDA226)
Plasmid pPICZalphaPDI eDA143 (reference citation)
 
 Parts amplified by PCR (using ProFlex PCR system, Applied Biosystems). Thermal Cycler

  
  
volumes in microliter
water up to 50Q5 HF enzyme0.7eDA223 100pg/uL1betaine 5x10447/4482.5/2.510mM dNTP (TAKARA)45xQ5 buffer10
   
 program Q5    Td-initial98oC30secTd98oC10sec33 cyclesTa55oC20secTe72oC1 min 40sec  Final extension72oC2 minhold4oChold
 
     447   (F)      PDI  part (MfeI)      CCCCAATTGACAAGCTTTTGATTTTAACG        448   (R)      PDI  part (NotI) introducing HIS-tag-TAA stop codon (italics)      TTTTTGCGGCCGCTTAATGATGATGGTGGTGATGCAACTCATCGTGAGCATCAGCTTC   
  
PCR clean-up    

RE digested with MfeI/NotI     
pPICZA Thermo Fisher vectors
 
12eDA223  nc 112ng/uL13PCR 447/44848MfeI-Hf (20u/ul)1.51NotI-hf (20u/ul)1.51rcutsmart86waterup to 80up to 60+ 7ul AP buffer + 2uL Antarctic Phosphatase
 60 uL of pPICZA MfeI/NotI digested mixed with 7 ul AP buffer and 2 uL     and incubation 30 min 
37 °C 

PCR product and pPICZA (MfeI/NotI) cleanup    
 
  Ligation reactions T4 DNA ligase (NEB)
   
 

   pUC-C 310/317pUC-C 310/31810 X buffer11HomC (310/317) B/K5.5HomC (310/318) B/K5.5pUC B/K11T4 DNA ligase11waterup to 10 ->
   

E.coli transformation (DH5alpha chemically LiAc competent cells)E.coli chemical competent cells
Selection on LB +100ug/mL   and growth at   37 °C 

Several colonies obtained on both agar plates. Single colonies transferred on new  LB +100ug/mL   plate, growth overnight and a single colonies submitted for bacterial colony PCR.
see protocol colony PCR
Material loaded on agarose gel for verification. 5 colonies verified positively using oligo 24/241.

   
 Positively verified clones re-cultured    in LB + 100 ug/ml carbenicillin, plasmid eDA226 was extracted by miniprep (plasmid preparation)   

SECTION: Preparation of triple-LHR integration array LHRA-PAOXPDI-LHRE-HygR-LHRZ
1.3. STEP 2 - preparation of semi-products, a partial ligated arrays 
 
            LHRA-Paox1PDI  LHRE-HygR-LHRZ      ml  ml    T7 DNA ligase  1  1    2x T7 ligase buffer  15  15    LHRA  5.7      Poax1PDI  8.3      LHRE    4    HygR-LHRZ    10    water      
 60 min 25 °C 

All ligation mixtures loaded on 1% agarose gel and separated to excise a corresponding size band
  
 DNA bands representing  expected size were excised and purified using   

DNA concentration calculated by DeNovix DS-11
 
            ng/uL  Molar concentration (nM)    LHRA-Paox1PDI  10  4    LHRE-HygR-LHRZ  16  7  
 

SECTION: Preparation of triple-LHR integration array LHRA-PAOXPDI-LHRE-HygR-LHRZ
1.4. pUC19 digestion with Esp3I
 Digestion with   

 
 
 
 1 2   33445 (test enzyme)6 (test enzyme)PCR  E 335/33846PCR D 304/30546PCR zeoC 308/318 (1)46PCR zeoC 308/318 (2)46PCR GFPcFH  302/303 (1)46PCR GFPcFH  302/303 (2)46puc19 nc22BsaI HF (20U/ul)20.5Esp3I (10U/ul))333330.5buf10x cutsmart66666666water 
 Digestion mixture cleanup   and DNA concentration checked on DeNovix DS-11 and 1%agarose gel
  
            ng/uL  Molar concentration (nM)    pUC19/Esp3I  35  19  
 
SECTION: Preparation of triple-LHR integration array LHRA-PAOXPDI-LHRE-HygR-LHRZ
1.5. STEP 3 - the final ligation of pre-assembled parts with Esp3I-linearized pUC19, E.coli transformation and colony PCR verification

   
            nM  ml    T4 DNA ligase    1    10x T4 ligase buffer    1.5    LHRA-Paox1PDI (4nM)  4  7    LHRE-HygR-LHRZ (7nM)   7  4    pUC19/Esp3I (19nM)  19  1.5    water    -  
 960 min 16 °C 

E.coli transformation (DH5alpha chemically LiAc competent cells)E.coli chemical competent cells
Selection on LB +100ug/mL +   and growth at   37 °C 

Several colonies obtained on both agar plates. Single colonies transferred on new  LB agar plates +100 ug/mL carbanicillin + 150 ug/mL hygromycin B
    

Growth overnight and a single colonies (8 colonies) submitted for bacterial colony PCR.
see protocol colony PCR
Material loaded on agarose gel for verification. 2 of 8 colonies verified positively using oligo 24/241. (expected 1.1kb amplicon) and 42/394 (0.91kb amplicon).
 
 
Colony 1 and 2 inoculated into LB + carbanicillin + hygromycin and growth   
Plasmids isolated by plasmid isolation (miniprep) Qiagen miniprep 60 min 
Sanger sequencing confirmed the sequence of eDA227

  

SECTION: Preparation of double-LHR integration array LHRE-PtefGFP-ZeoR-LHRZ
2. DNA parts preparation by PCR reactions LHRE+PtefGFP (1st pre-assembly ligation mixture)
List of DNA parts 
 
 
 
 
* eDA189 map .dna file   
**link https://www.protocols.io/view/preparation-of-parts-hygr-lhrz-and-zeor-lhrz-cqujvwun
SECTION: Preparation of double-LHR integration array LHRE-PtefGFP-ZeoR-LHRZ
2.1. STEP 1 - PCR amplification of parts LHRE(out)+PtefGFP and digestion with suitable IIS RE

Parts amplified by PCR (using ProFlex PCR system, Applied Biosystems). Thermal Cycler

  
  
volumes in microliter

 5xQ5 buffer10101010betaine 5x1010101010mM dNTP (TAKARA)4444335/3382.5/2.5304/3052.5/2.5308/3182.5/2.5302/3032.5/2.5eDA199 100ng/ul1eDA125 100ng/ul1eDA176 100ng/ul1eDA89 100ng/ul1Q5 HF enzyme0.70.70.70.7water up to 50
 
 program Q5    Td-initial98oC30secTd98oC10sec33 cyclesTa58oC20secTe72oC6 min   Final extension72oC1 minhold4oChold
  
All PCR products purified by   and analysed on 1% agarose gel (with ) 
      
 Digestion with    or   

 1 2   33445 (test enzyme)6 (test enzyme)PCR  E 335/33846PCR D 304/30546PCR zeoC 308/318 (1)46PCR zeoC 308/318 (2)46PCR GFPcFH  302/303 (1)46PCR GFPcFH  302/303 (2)46puc19 nc22BsaI HF (20U/ul)20.5Esp3I (10U/ul))333330.5buf10x cutsmart66666666water 
 Reaction 37 °C 360 min 
All PCR products purified by   and DNA concentration checked on DeNovix DS-11

DNA molar concentration calculation before ligation

  
 
            size (bp)  concentration (ng/ul)  Molar concentration (nM)      LHRE out  900  66  113    PtefGFP  1442  92  98    ZeoR-LHRZ  2171  111  72  
 
 

SECTION: Preparation of double-LHR integration array LHRE-PtefGFP-ZeoR-LHRZ
2.2. STEP 2 - preparation of semi-product, a partial ligated array LHRE-PtefGFP
 
 
 
            LHRA-Paox1PDI  LHRE-HygR-LHRZ      ml  ml    T7 DNA ligase  1  1    2x T7 ligase buffer  15  15    LHRA  5.7      Poax1PDI  8.3      LHRE    4    HygR-LHRZ    10    water      
 60 min 25 °C 
 Ligation mixture loaded on 1% agarose gel and separated to excise a corresponding size band
  
DNA band representing  expected size was excised and purified using   

DNA concentration calculated by DeNovix DS-11 
 
            size kbp  ng/uL  Molar   concentration (nM)    LHRA-PtefGDP  ~2.3  55  37      ZegR-LHRZ  2.17  101  72    

SECTION: Preparation of double-LHR  insertion array LHRE-PtefMCH-HygR-LHRZ
3.  DNA parts preparation by PCR reactions
List of DNA parts 
DNA partForward oligoRevers oligo producttemplateLHRE (OUT) (BsaI)F335-CGGTCTCCgggaGCGATCGCGACTTACCTCGTTTTAACTTAGTCGGR338-GGGTCTCCtgtcACAAGTTTAACTAAGCGTCAGCC900 bpeDA9PtefGFP (BsmBI)F302a-CCGTCTCCgacaAACATCCAAAGACGAAAGGTTGR304a-CCGTCTCCctacGCCTTCGAGCGTCCC1442 bpeDA189*ZeoR-LHRZ (BsmBI)F308-CCTGTCGACGGTCTCAgtagGACATGGAGGCCCAGAATACCCR318-GGGGTACCGGTCTCAcgcgGCGATCGCCCTGCAGGTTGAGTTGGCGAAGGTGCG2171 bpeDA105**
 * eDA191 map .dna file   
**link https://www.protocols.io/view/preparation-of-parts-hygr-lhrz-and-zeor-lhrz-cqujvwun


SECTION: Preparation of double-LHR  insertion array LHRE-PtefMCH-HygR-LHRZ
3.1. STEP 1 - PCR amplification of parts 
LHRE(out) (done in section 2, step 2.1) with oligos 335/338
HygR-LHRZ (done in section 1, step 1.2) with oligos 306/317

PtefMCh amplification and digestion with suitable IIS RE

Parts amplified by PCR (using ProFlex PCR system, Applied Biosystems). Thermal Cycler

  
  
volumes in microliter
 
 
 5xQ5 buffer10101010betaine 5x1010101010mM dNTP (TAKARA)4444335/3382.5/2.5304/3052.5/2.5308/3182.5/2.5302/3032.5/2.5eDA199 100ng/ul1eDA125 100ng/ul1eDA176 100ng/ul1eDA89 100ng/ul1Q5 HF enzyme0.70.70.70.7water up to 50
 
 program Q5    Td-initial98oC30secTd98oC10sec33 cyclesTa58oC20secTe72oC6 min   Final extension72oC1 minhold4oChold
  
PCR product purified by   and analysed on 1% agarose gel (with ) 
 
  
 
 Digestion with    or   

 
 
 
 1 2   33445 (test enzyme)6 (test enzyme)PCR  E 335/33846PCR D 304/30546PCR zeoC 308/318 (1)46PCR zeoC 308/318 (2)46PCR GFPcFH  302/303 (1)46PCR GFPcFH  302/303 (2)46puc19 nc22BsaI HF (20U/ul)20.5Esp3I (10U/ul))333330.5buf10x cutsmart66666666water 
 Reaction 37 °C 360 min 
All PCR products purified by   and DNA concentration checked on DeNovix DS-11

DNA molar concentration calculation before ligation

  
        size (bp)  concentration (ng/ul)  Molar   concentration (nM)      LHRE out  900  66  48  113    PtefGFP  1442  92  34  98    ZeoR-LHRZ  2171  101  60  72  

SECTION: Preparation of double-LHR  insertion array LHRE-PtefMCH-HygR-LHRZ
3.3. STEP 3 - the final ligation of pre-assembled parts with Esp3I-linearized pUC19, E.coli transformation and colony PCR verification
 
            uL  nM    T4 DNA ligase  1      10x T4 ligase buffer  1      LHRE-PtefMCH  1.6  64    ZeoR-LHRZ  1.4  72    pUC19/Esp3I  5  19  
 
  960 min 16 °C 

E.coli transformation (DH5alpha chemically LiAc competent cells)E.coli chemical competent cells
Selection on LB +100ug/mL carbanicillin + 150ug/mL hygromycin B
  
  
growth at   37 °C 
 
Several fluorescent colonies obtained on both LB agar plates. Single colonies transferred on new  LB agar plates +100 ug/mL carbanicillin + 50 ug/mL zeocin 
 

Single fluorescent colonies inoculated on fresh LB + carbanicillin/hygromycin and growth   
Next day a pasmid isolation (miniprep) Qiagen miniprep 60 min 
Sanger sequencing confirmed the sequence of eDA199
  

SECTION: Preparation of double-LHR  insertion array LHRE-PtefMCH-HygR-LHRZ
3.2. STEP 2 - preparation of semi-product, a partial ligated array LHRE-PtefMCh
 

 
            LHRA-Paox1PDI  LHRE-HygR-LHRZ      ml  ml    T7 DNA ligase  1  1    2x T7 ligase buffer  15  15    LHRA  5.7      Poax1PDI  8.3      LHRE    4    HygR-LHRZ    10    water      
 60 min 25 °C 
 Ligation mixture loaded on 1% agarose gel and separated to excise a corresponding size band
  
DNA band representing  expected size was excised and purified using   

DNA concentration calculated by DeNovix DS-11 
 
 
            size kbp  ng/uL  Molar concentration (nM)    LHRE-PtefMCH  ~2.3  96  64    HygR-LHRZ  2.5  101  72  
 * HygR-LHRZ from step 1.2 
SECTION: Preparation of double-LHR integration array LHRE-PtefGFP-ZeoR-LHRZ
2.3. STEP 3 - the final ligation of pre-assembled parts with Esp3I-linearized pUC19, E.coli transformation and colony PCR verification
 
 
            uL  nM    T4 DNA ligase  1      10x T4 ligase buffer  1      LHRE-PtefGFP  2.3  37    ZeoR-LHRZ  1.2  72    pUC19/Esp3I  4.5  19  
 
  960 min 16 °C 

E.coli transformation (DH5alpha chemically LiAc competent cells)E.coli chemical competent cells
Selection on LB +100ug/mL carbanicillin + 50ug/mL zeocin
     growth at   37 °C 

  
Several fluorescent colonies obtained on both agar plates. Single colonies transferred on new  LB agar plates +100 ug/mL carbanicillin + 50 ug/mL zeocin 
 

Single fluorescent colonies inoculated on fresh LB + carbanicillin/zeocin and growth   
Next day a pasmid isolation (miniprep) Qiagen miniprep 60 min 
Sanger sequencing confirmed the sequence of eDA197
  
 
SECTION: Preparation of triple-LHR insertion array LHRA-GFP:CFH-LHRE-ZeoR-LHRZ
4. DNA parts preparation by PCR reactions
List of DNA parts 
 
 
* map    plasmid prepared as described in the paper (DOI.....PBarlow,DAbramczyk)
**link https://www.protocols.io/view/preparation-of-parts-hygr-lhrz-and-zeor-lhrz-cqujvwun

SECTION: Preparation of triple-LHR insertion array LHRA-GFP:CFH-LHRE-ZeoR-LHRZ
4.1. STEP 1 - PCR amplification of parts and digestion with suitable IIS RE

Parts amplified by PCR (using ProFlex PCR system, Applied Biosystems). Thermal Cycler

  
  
volumes in microliters
 

 
 5xQ5 buffer10101010betaine 5x1010101010mM dNTP (TAKARA)4444335/3382.5/2.5304/3052.5/2.5308/3182.5/2.5302/3032.5/2.5eDA199 100ng/ul1eDA125 100ng/ul1eDA176 100ng/ul1eDA89 100ng/ul1Q5 HF enzyme0.70.70.70.7water up to 50
 Part LHRE (out) generated as described above in protocol 2.1
Part ZeoR-LHRZ generated as described above in protocol 2.1

 program Q5    Td-initial98oC30secTd98oC10sec33 cyclesTa58oC20secTe72oC6 min   Final extension72oC1 minhold4oChold
  
All PCR products purified by   and analysed on 1% agarose gel (with ) 
 
  
  

 Digestion with    or   

 
 
 
 1 2   33445 (test enzyme)6 (test enzyme)PCR  E 335/33846PCR D 304/30546PCR zeoC 308/318 (1)46PCR zeoC 308/318 (2)46PCR GFPcFH  302/303 (1)46PCR GFPcFH  302/303 (2)46puc19 nc22BsaI HF (20U/ul)20.5Esp3I (10U/ul))333330.5buf10x cutsmart66666666water 
 All PCR products purified by   and DNA concentration checked on DeNovix DS-11

DNA molar concentration calculation before ligati

              size (bp)  concentration (ng/ul)  Molar concentration (nM)    LHRE out  900  66  48    PaoxGFPCFH  5991  123  34    LHRD  1105  120  142    ZeoR-LHRZ  2171  101  72  
  
SECTION: Preparation of triple-LHR insertion array LHRA-GFP:CFH-LHRE-ZeoR-LHRZ
4.2. STEP 2 - preparation of semi-products, a partial ligated arrays 
 
 
 
            LHRA-Paox1PDI  LHRE-HygR-LHRZ      ml  ml    T7 DNA ligase  1  1    2x T7 ligase buffer  15  15    LHRA  5.7      Poax1PDI  8.3      LHRE    4    HygR-LHRZ    10    water      
 60 min 25 °C 

All ligation mixtures loaded on 1% agarose gel and separated to excise a corresponding size band

  
DNA band representing  expected size was excised and purified using   

DNA concentration calculated by DeNovix DS-11 
  
   
            ng/uL  Molar concentration (nM)    LHRE-PaoxGFPCFH  22  5    LHRD-ZeoR-LHRZ  9  4  
 
SECTION: Preparation of triple-LHR insertion array LHRA-GFP:CFH-LHRE-ZeoR-LHRZ
4.3. STEP 3 - the final ligation of pre-assembled parts with Esp3I-linearized pUC19, E.coli transformation and colony PCR verification

 
 nMmlT4 DNA ligase110x T4 ligase buffer1.5LHRE-PaoxGFPCFH55.0LHRD-ZeoR-LHRZ 46.2pUC19/Esp3I (19nM)191.3water
  960 min 16 °C 

E.coli transformation (DH5alpha chemically LiAc competent cells)E.coli chemical competent cells
Selection on LB +100ug/mL + 50ug/mL
 
  

 and growth at   37 °C 

 

 
  

Several colonies obtained on both agar plates. Single colonies transferred on new  LB agar plates +100 ug/mL carbanicillin + 50 ug/mL zeocin B.
Two of them submitted for bacterial colony PCR.
see protocol colony PCR
 
352/353274/278355/354345/40351/133374/370 puc-E 0.45kbGFP-FH 0.75kb Aox-D 0.29kbD-zeo 0.9kbzeoc-C 0.6kb0.7kb contr+  1clone1 (eDA250)OKOKOKOKOKOK2clone2nonenonepoornonenonenone
  
Material loaded on agarose gel for verification. 1 of 2 colonies verified positively verified

The colony inoculated on fresh LB + carbanicillin/zeocin and growth   
Next day a pasmid isolation (miniprep) Qiagen miniprep 60 min 
Sanger sequencing confirmed the sequence of eDA250
  

Plasmidsaurus sequencing confirms the correct sequence (added to .dna file)