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Supplementary Figure 1

Derivation of pBAC2015.

(a) pBAC2015 was derived from pBACe3.6 by insertion of oriV and removal of a 30 bp repeat and the loxP site. (b) Comparison of direct cloning with pBACe3.6 and pBAC2015. Homology arms to the 37.5 kb Photorhabdus luminescens plu3535-plu3532 gene cluster were added using the method illustrated in Supplementary Figure 2. Most of the colonies obtained with pBACe3.6 were due to intramolecular recombination through the 30 bp repeat. Its removal in pBAC2015 dramatically reduced the unwanted background so that most colonies contained the intended product.

Nature Protocols: doi:10.1038/nprot.2016.054


Construction of linear BAC direct cloning vectors.

(a) The cassette containing pBR322, amp and ccdB is amplified by two rounds of PCR to attach homology arms to the gene cluster (ha1 and ha2) and pBAC2015 (ha3 and ha4) respectively. (b) The cassette containing pBR322, amp and ccdB is recombined with pBAC2015 by LLHR in RecET expressing CcdB resistant strain, E. coli GBdir-gyrA462. Because the pBR322-amp-ccdB-rpsL plasmid carries the rpsL gene, in addition to ampicillin and chloramphenicol selection, streptomycin selection can be added to eliminate carryover background from the PCR template. The BAC vector is linearized by BamHI digestion to expose the terminal homology arms.



Optimization of R6K plasmids with transposition cassette for different hosts.

(a) pR6K-oriT-tnpA-genta is the same as pR6K-oriT-tnpA-kan except the gentamicin resistance gene replaced the kanamycin resistance gene. (b) pR6K-oriT-tnpA-apra is the same as pR6K-oriT-tnpA-kan except for the inclusion of the apramycin resistance gene and the NcoI site. (c) Exchanging the promoter of tnpA via LLHR. Electroporate the BstZ17I digested pR6K-oriT-TnpA-kan plasmid together with a synthetic oligonucleotide, a synthetic DNA or a PCR product into RecET expressing pir strain, E. coli GBdir-pir116, for LLHR. In case of using synthetic oligonucleotides, the homology arms can be as short as 20 nt to ensure the oligonucleotide is not longer than 140 nt.


1

Supplementary Table 1 E. coli strains used in this protocol.

Strains Relevant features Use Reference

GB05RedTrfA DH10B, fhuA::IS2, ∆ybcC, ∆recET, PRhaSR-γβαA, PBAD-trfA

Linear plus circular homologous recombination Replication of oriVRK2 containing plasmid

1

GBdir-gyrA462 DH10B, fhuA::IS2, ∆ybcC, ∆recET, PBAD-ETγA, gyrAArg462Cys

Linear plus linear homologous recombination Replication of ccdB containing plasmid

This study

GBdir-pir116 DH10B, fhuA::IS2, ∆ybcC, ∆recET, pir116

Linear plus linear homologous recombination Replication of R6K plasmid

This study


2

Supplementary Table 2 Plasmids used in this protocol.

Plasmids Relevant features and use Sequence References

pSC101-BAD-ETgA-tet RecET expression plasmid for linear plus linear homologous recombination

Supplementary note 1 2

pBR322-amp-tetR-tetO-hyg-ccdB PCR template to generate a linear vector for direct cloning Supplementary note 2 This study

p15A-cm-tetR-tetO-hyg-ccdB PCR template to generate a linear vector for direct cloning Supplementary note 3 This study

pBAC2015 Copy-control BAC vector without repetitive sequences Supplementary note 4 This study

pR6K-oriT-TnpA-kan Suicide plasmid to release a conjugation-transposition cassette to modify a cloning vector (PCR free recombineering)

Supplementary note 5 This study

pR6K-oriT-TnpA-apra Suicide plasmid to release a conjugation-transposition cassette to modify a cloning vector (PCR free recombineering)


pR6K-oriT-TnpA-genta Suicide plasmid to release a conjugation-transposition cassette to modify a cloning vector (PCR free recombineering)


pR6K-oriT-phiC31 Suicide plasmid to release a conjugation-site-specific-integration cassette to modify a cloning vector (PCR free recombineering)


pBBR1-kan-hyg-ccdB Suicide plasmid to release a linear broad-host-range vector for subcloning (PCR free recombineering)


pBR322-amp-ccdB-rpsL PCR template of pBR322-amp-ccdB for pBAC2015 modification to introduce homology sequences for direct cloning



3

Supplementary Table 3 Efficiency of direct cloning of PKS-NRPS gene clusters from Photorhabdus luminescens.

Gene cluster Size

(bp)

Restriction fragment

(bp)

5’ to homology 3’ to homology Vector Colony number

( /ml )

Correct / checked

plu3535-plu3532 32,886 37,501 0 0 pBR322-amp-tetR-tetO 1,158 ± 138 8/12

p15A-cm-tetR-tetO 580 ± 10 12/12

pBAC2015 20 ± 5 6/10

plu1113-plu1115 21,732 29,834 7,181 0 pBR322-amp-tetR-tetO 265 ± 18 12/12


4

Supplementary Method 1: Preparation of genomic DNA from bacteria for direct cloning ●TIMING 4-6 h

1. Harvest bacterial cells from 50 ml fresh culture in stationary phase by centrifugation at 8,300g

for 5 min, room temperature (RT).

2. Aspirate away the supernatant as much as possible.

3. Resuspend the cells thoroughly in 30 ml autoclaved ddH2O by vortexing at maximium speed.

4. Spin down at 8,300g for 5 min, RT.

5. Aspirate away the supernatant as much as possible.

6. Prepare the cell lysis by using option A for Gram-negative bacteria, or option B for

Gram-positive bacteria.

(A) lysis of Gram-negative bacteria

(i) Resuspend the cells thoroughly in 8 ml 10 mM Tris-Cl, pH 8.0.

(ii) Add 500 µl 20 mg ml-1 proteinase K and invert to mix, then add 1 ml 10% SDS and invert

to mix.

(iii) Incubate at 50 °C for 2 h in water bath with occasionally inverting until the solution become

clear.

(iv) Add 10 ml Phenol-chloroform-isoamyl alcohol mixture (25:24:1) and mix thoroughly by

inversion to create an emulsion.

(B) lysis of Gram-positive bacteria

(i) Resuspend the cells thoroughly in 8 ml SET (75 mM NaCl, 25 mM EDTA, 20 mM Tris, pH

8.0) by vortexing.

(ii) Add 10 mg lysozyme, invert to mix and incubate at 37 °C for 1 h in water bath with

occasionally inverting.

(iii) Add 500 µl 20 mg ml-1 proteinase K and invert to mix, and then add 1 ml 10% SDS and

invert to mix.

(iv) Incubate at 50 °C for 2 h in water bath with occasionally inverting until the solution become

clear.

(v) Add 3.5 ml 5 M NaCl and invert to mix.

(vi) Add 15 ml Phenol-chloroform-isoamyl alcohol mixture (25:24:1) and mix thoroughly by

inversion to create an emulsion.

7. Centrifuge at 8,300g for 30 min, RT.

8. Transfer 500 µl of the aqueous phase (top layer) gently by using an end-cut wide-bore 1-ml tip

into a 2-ml microcentrifuge tube.

9. Add 35 µl 3 M sodium acetate (pH 7.5) and gently invert to mix.

10. Add 1.2 ml absolute ethanol and gently invert to mix.


5

11. Prepare 2-ml microcentrifuge tubes with 1.5 ml 70% ethanol and transfer cotton-like DNA into

these tubes by using a yellow tip.

12. Pellet the genomic DNA with centrifuge at 9,400g for 1 min, RT.

13. Discard the supernatant, invert the tube on a paper towel to let all of the liquid drain off the

pellet.

▲ CRITICAL STEP Care must be taken while discarding the supernatant because the DNA pellet

can slip away from the tube wall.

14. Dry the pellet at RT on bench for about 30 min until no liquid is left. The white DNA pellet will

become transparent when it is dry.

15. Resuspend the DNA pellet in 500 µl 10 mM Tris-Cl, pH 8.0. The concentration of genomic is

~200 ng ul-1.

Supplementary References

1. Nedelkova, M. et al. Targeted isolation of cloned genomic regions by recombineering for

haplotype phasing and isogenic targeting. Nucleic Acids Res. 39, e137, (2011).

2. Fu, J. et al. Full-length RecE enhances linear-linear homologous recombination and facilitates

direct cloning for bioprospecting. Nat. Biotechnol. 30, 440-446, (2012).


Supplementary Note 1: Complete sequence of the pSC101-BAD-ETgA-tet plasmid in Genbank

format.

LOCUS pSC101-BAD-ETgA-tet 9233 bp DNA circular SYN 27-Jun-2015

DEFINITION pSC101-BAD-ETgA-tet

ACCESSION pSC101-BAD-ETgA-tet

KEYWORDS

SOURCE

ORGANISM

FEATURES Location/Qualifiers

rep_origin 1..362

/label="pSC101 ori"

/note="replication origin"

CDS 746..1696

/label="repA"

/note="replication protein, temperature sensitive"

CDS 1781..2971

/label="tet"

/note="tetracycline resistance gene"

CDS complement(3106..3984)

/label="araC"

/note="regulator gene of L-arabinose operon"

promoter 4012..4260

/label="BAD promoter"

CDS 4330..6930

/label="recE"

/note="exonuclease from Rac prophage"

CDS 6923..7732

/label="recT"

/note="ssDNA annealing protein, recombinase from Rac prophage"

CDS 7735..8151

/label="gam"

/note="lambda Red gam protein, recBCD inhibitor"

CDS 8154..9215

/label="recA"

/note="E. coli RecA protein"

ORIGIN

1 gtaagacggg taagcctgtt gatgataccg ctgccttact gggtgcatta gccagtctga

61 atgacctgtc acgggataat ccgaagtggt cagactggaa aatcagaggg caggaactgc

121 tgaacagcaa aaagtcagat agcaccacat agcagacccg ccataaaacg ccctgagaag

181 cccgtgacgg gcttttcttg tattatgggt agtttccttg catgaatcca taaaaggcgc

241 ctgtagtgcc atttaccccc attcactgcc agagccgtga gcgcagcgaa ctgaatgtca

301 cgaaaaagac agcgactcag gtgcctgatg gtcggagaca aaaggaatat tcagcgattt


361 gcccgagctt gcgagggtgc tacttaagcc tttagggttt taaggtctgt tttgtagagg

421 agcaaacagc gtttgcgaca tccttttgta atactgcgga actgactaaa gtagtgagtt

481 atacacaggg ctgggatcta ttctttttat ctttttttat tctttcttta ttctataaat

541 tataaccact tgaatataaa caaaaaaaac acacaaaggt ctagcggaat ttacagaggg

601 tctagcagaa tttacaagtt ttccagcaaa ggtctagcag aatttacaga tacccacaac

661 tcaaaggaaa aggactagta attatcattg actagcccat ctcaattggt atagtgatta

721 aaatcaccta gaccaattga gatgtatgtc tgaattagtt gttttcaaag caaatgaact

781 agcgattagt cgctatgact taacggagca tgaaaccaag ctaattttat gctgtgtggc

841 actactcaac cccacgattg aaaaccctac aaggaaagaa cggacggtat cgttcactta

901 taaccaatac gctcagatga tgaacatcag tagggaaaat gcttatggtg tattagctaa

961 agcaaccaga gagctgatga cgagaactgt ggaaatcagg aatcctttgg ttaaaggctt

1021 tgagattttc cagtggacaa actatgccaa gttctcaagc gaaaaattag aattagtttt

1081 tagtgaagag atattgcctt atcttttcca gttaaaaaaa ttcataaaat ataatctgga

1141 acatgttaag tcttttgaaa acaaatactc tatgaggatt tatgagtggt tattaaaaga

1201 actaacacaa aagaaaactc acaaggcaaa tatagagatt agccttgatg aatttaagtt

1261 catgttaatg cttgaaaata actaccatga gtttaaaagg cttaaccaat gggttttgaa

1321 accaataagt aaagatttaa acacttacag caatatgaaa ttggtggttg ataagcgagg

1381 ccgcccgact gatacgttga ttttccaagt tgaactagat agacaaatgg atctcgtaac

1441 cgaacttgag aacaaccaga taaaaatgaa tggtgacaaa ataccaacaa ccattacatc

1501 agattcctac ctacgtaacg gactaagaaa aacactacac gatgctttaa ctgcaaaaat

1561 tcagctcacc agttttgagg caaaattttt gagtgacatg caaagtaagc atgatctcaa

1621 tggttcgttc tcatggctca cgcaaaaaca acgaaccaca ctagagaaca tactggctaa

1681 atacggaagg atctgatctc atgtttgaca gcttatcatc gataagcttt aatgcggtag

1741 tttatcacag ttaaattgct aacgcagtca ggcaccgtgt atgaaatcta acaatgcgct

1801 catcgtcatc ctcggcacca tcaccctgga tgctgtaggc ataggcttgg ttatgccggt

1861 actgccgggc ctcttgcggg atatcgtcca ttccgacagc atcgccagtc actatggcgt

1921 gctgctagcg ctatatgcgt tgatgcaatt tctatgcgca cccgttctcg gagcactgtc

1981 cgaccgcttt ggccgccgcc cagtcctgct cgcttcgcta cttggagcca ctatcgacta

2041 cgcgatcatg gcgaccacac ccgtcctgtg gatcctctac gccggacgca tcgtggccgg

2101 catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga

2161 agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg

2221 ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc

2281 ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg

2341 agagcgtcga ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg

2401 gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca

2461 ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat

2521 gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac

2581 tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggccga

2641 cgcgctgggc tacgtcttgc tggcgttcgc gacgcgaggc tggatggcct tccccattat

2701 gattcttctc gcttccggcg gcatcgggat gcccgcgttg caggccatgc tgtccaggca

2761 ggtagatgac gaccatcagg gacagcttca aggatcgctc gcggctctta ccagcctaac

2821 ttcgatcatt ggaccgctga tcgtcacggc gatttatgcc gcctcggcga gcacatggaa

2881 cgggttggca tggattgtag gcgccgccct ataccttgtc tgcctccccg cgttgcgtcg

2941 cggtgcatgg agccgggcca cctcgacctg aatggaagcc ggcggcacct cgctaacgga


3001 ttcaccactc caagatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac

3061 cctatgctac tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac

3121 ggctacatca ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt

3181 gcatttttta aatacccgcg agaaatagag ttgatcgtca aaaccaacat tgcgaccgac

3241 ggtggcgata ggcatccggg tggtgctcaa aagcagcttc gcctggctga tacgttggtc

3301 ctcgcgccag cttaagacgc taatccctaa ctgctggcgg aaaagatgtg acagacgcga

3361 cggcgacaag caaacatgct gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg

3421 atcgctgatg tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc

3481 gttaatcgct tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg ccagcagctc

3541 cgaatagcgc ccttcccctt gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg

3601 cggctggtgc gcttcatccg ggcgaaagaa ccccgtattg gcaaatattg acggccagtt

3661 aagccattca tgccagtagg cgcgcggacg aaagtaaacc cactggtgat accattcgcg

3721 agcctccgga tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc

3781 cggtcggcaa acaaattctc gtccctgatt tttcaccacc ccctgaccgc gaatggtgag

3841 attgagaata taacctttca ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt

3901 ggcctcaatc ggcgttaaac ccgccaccag atgggcatta aacgagtatc ccggcagcag

3961 gggatcattt tgcgcttcag ccatactttt catactcccg ccattcagag aagaaaccaa

4021 ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac

4081 caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca aagccatgac

4141 aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg attatttgca

4201 cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg atcctacctg

4261 acgcttttta tcgcaactct ctactgtttc tccatacccg tttttttggg ctagcaggag

4321 gaattcacta tgagcacaaa accactcttc ctgttacgga aagcgaaaaa atcatccggt

4381 gaacctgacg tcgtcctgtg ggcaagcaac gattttgaat cgacctgtgc cactctggac

4441 tacctgatcg ttaagtcagg taaaaaactg agcagctatt ttaaagctgt tgccacgaat

4501 tttcctgtcg ttaatgacct gcccgctgaa ggtgagatcg attttacctg gagtgaacgc

4561 tatcaactca gcaaagactc catgacatgg gaactaaaac cgggagcagc accagacaac

4621 gctcactatc aaggcaatac caacgtcaac ggcgaagaca tgactgagat tgaggagaat

4681 atgctactcc caatttctgg ccaggaactg cccattcgtt ggcttgctca acacggcagc

4741 gaaaaaccgg taacgcacgt ttcacgcgac ggactccagg cattacacat tgctcgggct

4801 gaagaactac cggctgttac tgccctggct gtttcccaca aaaccagcct gctcgacccg

4861 ctggaaattc gcgaactcca caaactggtt cgtgacactg acaaagtttt ccctaatcct

4921 ggtaattcaa acctgggact gataactgct tttttcgaag catacctgaa cgctgactac

4981 accgatcgag gactgctgac aaaagagtgg atgaagggta atcgtgtttc acacatcact

5041 cgcacggctt ccggtgctaa tgctggcggc ggaaacctca ccgatcgcgg cgaaggtttc

5101 gtacacgatc tgacgtcact ggcgcgcgac gtagccactg gcgtactggc ccgttcaatg

5161 gatctggaca tctataacct tcatccggca cacgctaaac gcattgagga aattatcgct

5221 gaaaataaac cgcccttttc tgttttccgc gacaaattca tcaccatgcc tggcgggctg

5281 gattattccc gcgccatcgt ggttgcgtcc gtaaaagaag caccaattgg gatcgaggtc

5341 atccccgcgc acgtcactga atatctgaac aaagtactga ctgaaaccga tcatgccaac

5401 cctgatccgg aaatcgtgga tattgcctgc ggtcgctcct ctgccccgat gccgcagcga

5461 gtaacagaag aaggaaaaca ggatgatgaa gaaaaaccgc aaccatctgg aacaacggca

5521 gttgaacagg gagaggctga aacaatggaa ccggacgcaa ctgaacatca tcaggacacg

5581 cagccgctgg atgctcagtc acaggtaaat tctgttgatg cgaaatatca ggaactgcgg


5641 gcagaactcc atgaagcccg gaaaaacatt ccatcaaaaa atcctgtcga tgacgataaa

5701 ttgcttgctg catcacgtgg tgaatttgtt gacggaatta gcgacccgaa cgatccgaaa

5761 tgggtaaagg ggatccagac tcgcgattgt gtgtaccaga accagccaga aacggaaaaa

5821 accagcccag atatgaatca acctgagcca gtagtgcaac aggaaccgga aatagcctgc

5881 aatgcctgcg gccagactgg cggggataac tgccctgact gtggtgcggt gatgggcgac

5941 gcaacatacc aggaaacatt cgatgaagag agtcaggttg aagctaagga aaatgatccg

6001 gaggaaatgg aaggcgctga acatccgcac aatgagaatg ctggcagcga tccgcatcgc

6061 gattgcagtg atgaaactgg cgaagtcgca gatcccgtaa tcgtagaaga catagagcca

6121 ggtatttatt acggaatttc gaatgagaat taccacgcgg gtcccggtat cagtaagtct

6181 cagctcgatg acattgctga tactccggca ctatatttgt ggcgtaaaaa tgcccccgtg

6241 gacaccacaa agacaaaaac gctcgattta ggaactgctt tccactgccg ggtacttgaa

6301 ccggaagaat tcagtaaccg ctttatcgta gcacctgaat ttaaccgccg tacaaacgcc

6361 ggaaaagaag aagagaaagc gtttctgatg gaatgcgcaa gcacaggaaa aacggttatc

6421 actgcggaag aaggccggaa aattgaactc atgtatcaaa gcgttatggc tttgccgctg

6481 gggcaatggc ttgttgaaag cgccggacac gctgaatcat caatttactg ggaagatcct

6541 gaaacaggaa ttttgtgtcg gtgccgtccg gacaaaatta tccctgaatt tcactggatc

6601 atggacgtga aaactacggc ggatattcaa cgattcaaaa ccgcttatta cgactaccgc

6661 tatcacgttc aggatgcatt ctacagtgac ggttatgaag cacagtttgg agtgcagcca

6721 actttcgttt ttctggttgc cagcacaact attgaatgcg gacgttatcc ggttgaaatt

6781 ttcatgatgg gcgaagaagc aaaactggca ggtcaacagg aatatcaccg caatctgcga

6841 accctgtctg actgcctgaa taccgatgaa tggccagcta ttaagacatt atcactgccc

6901 cgctgggcta aggaatatgc aaatgactaa gcaaccacca atcgcaaaag ccgatctgca

6961 aaaaactcag ggaaaccgtg caccagcagc agttaaaaat agcgacgtga ttagttttat

7021 taaccagcca tcaatgaaag agcaactggc agcagctctt ccacgccata tgacggctga

7081 acgtatgatc cgtatcgcca ccacagaaat tcgtaaagtt ccggcgttag gaaactgtga

7141 cactatgagt tttgtcagtg cgatcgtaca gtgttcacag ctcggacttg agccaggtag

7201 cgccctcggt catgcatatt tactgccttt tggtaataaa aacgaaaaga gcggtaaaaa

7261 gaacgttcag ctaatcattg gctatcgcgg catgattgat ctggctcgcc gttctggtca

7321 aatcgccagc ctgtcagccc gtgttgtccg tgaaggtgac gagtttagct tcgaatttgg

7381 ccttgatgaa aagttaatac accgcccggg agaaaacgaa gatgccccgg ttacccacgt

7441 ctatgctgtc gcaagactga aagacggagg tactcagttt gaagttatga cgcgcaaaca

7501 gattgagctg gtgcgcagcc tgagtaaagc tggtaataac gggccgtggg taactcactg

7561 ggaagaaatg gcaaagaaaa cggctattcg tcgcctgttc aaatatttgc ccgtatcaat

7621 tgagatccag cgtgcagtat caatggatga aaaggaacca ctgacaatcg atcctgcaga

7681 ttcctctgta ttaaccgggg aatacagtgt aatcgataat tcagaggaat aagtatggat

7741 attaatactg aaactgagat caagcaaaag cattcactaa ccccctttcc tgttttccta

7801 atcagcccgg catttcgcgg gcgatatttt cacagctatt tcaggagttc agccatgaac

7861 gcttattaca ttcaggatcg tcttgaggct cagagctggg cgcgtcacta ccagcagctc

7921 gcccgtgaag agaaagaggc agaactggca gacgacatgg aaaaaggcct gccccagcac

7981 ctgtttgaat cgctatgcat cgatcatttg caacgccacg gggccagcaa aaaatccatt

8041 acccgtgcgt ttgatgacga tgttgagttt caggagcgca tggcagaaca catccggtac

8101 atggttgaaa ccattgctca ccaccaggtt gatattgatt cagaggtata aatatggcta

8161 tcgacgaaaa caaacagaaa gcgttggcgg cagcactggg ccagattgag aaacaatttg

8221 gtaaaggctc catcatgcgc ctgggtgaag accgttccat ggatgtggaa accatctcta


8281 ccggttcgct ttcactggat atcgcgcttg gggcaggtgg tctgccgatg ggccgtatcg

8341 tcgaaatcta cggaccggaa tcttccggta aaaccacgct gacgctgcag gtgatcgccg

8401 cagcgcagcg tgaaggtaaa acctgtgcgt ttatcgatgc tgaacacgcg ctggacccaa

8461 tctacgcacg taaactgggc gtcgatatcg acaacctgct gtgctcccag ccggacaccg

8521 gcgagcaggc actggaaatc tgtgacgccc tggcgcgttc tggcgcagta gacgttatcg

8581 tcgttgactc cgtggcggca ctgacgccga aagcggaaat cgaaggcgaa atcggcgact

8641 ctcacatggg ccttgcggca cgtatgatga gccaggcgat gcgtaagctg gcgggtaacc

8701 tgaagcagtc caacacgctg ctgatcttca tcaaccagat ccgtatgaaa attggtgtga

8761 tgttcggtaa cccggaaacc actaccggtg gtaacgcgct gaaattctac gcctctgttc

8821 gtctcgacat ccgtcgtatc ggcgcggtga aagagggcga aaacgtggtg ggtagcgaaa

8881 cccgcgtgaa agtggtgaag aacaaaatcg ctgcgccgtt taaacaggct gaattccaga

8941 tcctctacgg cgaaggtatc aacttctacg gcgaactggt tgacctgggc gtaaaagaga

9001 agctgatcga gaaagcaggc gcgtggtaca gctacaaagg tgagaagatc ggtcagggta

9061 aagcgaatgc gactgcctgg ctgaaagata acccggaaac cgcgaaagag atcgagaaga

9121 aagtacgtga gttgctgctg agcaacccga actcaacgcc ggatttctct gtagatgata

9181 gcgaaggcgt agcagaaact aacgaagatt tttaagcttc tcgagtctag aca

//


Supplementary Note 2: Complete sequence of the p15A-cm-tetR-tetO-hyg-ccdB plasmid

in Genbank format.

LOCUS p15A-cm-tetR-tetO-hyg-ccdB 4227 bp DNA circular SYN 27-Jun-2015

DEFINITION p15A-cm-tetR-tetO-hyg-ccdB

ACCESSION p15A-cm-tetR-tetO-hyg-ccdB

KEYWORDS

SOURCE

ORGANISM


rep_origin 1..718

/label="p15A ori"



/label="cm"

/note="chloramphenicol resistance gene"


/label="tetR"

/note="tetO repressor gene"

promoter 2350..2431

/label="tetO promoter"

primer-reverse complement(2432..2454)

/label="PCR primer"

CDS 2676..3701

/label="hyg"

/note="hygromycin resistance gene"

CDS 3750..4055

/label="ccdB"

/note="CcdB toxic protein"

primer 4062..4084

/label="PCR primer"

ORIGIN

1 cctagggata tattccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg

61 gcgagcggaa atggcttacg aacggggcgg agatttcctg gaagatgcca ggaagatact

121 taacagggaa gtgagagggc cgcggcaaag ccgtttttcc ataggctccg cccccctgac

181 aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa acccgacagg actataaaga

241 taccaggcgt ttccccctgg cggctccctc gtgcgctctc ctgttcctgc ctttcggttt

301 accggtgtca ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga cactcagttc

361 cgggtaggca gttcgctcca agctggactg tatgcacgaa ccccccgttc agtccgaccg

421 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gaaagacatg caaaagcacc

481 actggcagca gccactggta attgatttag aggagttagt cttgaagtca tgcgccggtt

541 aaggctaaac tgaaaggaca agttttggtg actgcgctcc tccaagccag ttacctcggt

601 tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt


661 tttcagagca agagattacg cgcagaccaa aacgatctca agaagatcat cttattaatc

721 agataaaata tttctagatt tcagtgcaat ttatctcttc aaatgtagca cctgaagtca

781 gccccatacg atataagttg ttactagtgc ttggattctc accaataaaa aacgcccggc

841 ggcaaccgag cgttctgaac aaatccagat ggagttctga ggtcattact ggatctatca

901 acaggagtcc aagcgagctc gatatcaaat tacgccccgc cctgccactc atcgcagtac

961 tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc

1021 tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catggtgaaa

1081 acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt gaaactcacc

1141 cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa ataggccagg

1201 ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg

1261 tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa

1321 gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg aaattccgga

1381 tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt gtgcttattt

1441 ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt ataggtacat

1501 tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga tatatcaacg

1561 gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga aaatctcgat

1621 aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt ggaacctctt

1681 acgtgccgat caacgtctca ttttcgccag atatcgacgt cttaagaccc actttcacat

1741 ttaagttgtt tttctaatcc gcatatgatc aattcaaggc cgaataagaa ggctggctct

1801 gcaccttggt gatcaaataa ttcgatagct tgtcgtaata atggcggcat actatcagta

1861 gtaggtgttt ccctttcttc tttagcgact tgatgctctt gatcttccaa tacgcaacct

1921 aaagtaaaat gccccacagc gctgagtgca tataatgcat tctctagtga aaaaccttgt

1981 tggcataaaa aggctaattg attttcgaga gtttcatact gtttttctgt aggccgtgta

2041 cctaaatgta cttttgctcc atcgcgatga cttagtaaag cacatctaaa acttttagcg

2101 ttattacgta aaaaatcttg ccagctttcc ccttctaaag ggcaaaagtg agtatggtgc

2161 ctatctaaca tctcaatggc taaggcgtcg agcaaagccc gcttattttt tacatgccaa

2221 tacaatgtag gctgctctac acctagcttc tgggcgagtt tacgggttgt taaaccttcg

2281 attccgacct cattaagcag ctctaatgcg ctgttaatca ctttactttt atctaatcta

2341 gacatcatta attcctaatt tttgttgaca ctctatcgtt gatagagtta ttttaccact

2401 ccctatcagt gatagagaaa agaattcaaa agatctaaag aggagaaagg atctctcgag

2461 gacataagcc tgttcggttc gtaaactgta ctgcaagtag cgtatgcgct cacgcaactg

2521 gtccagaacc ttgaccgaac gcagcggtgg taacggcgca gtggcggttt tcatggcttg

2581 ttatgactgt ttttttgtac agtctatgcc tcgggcatcc aagcagcaag cgcgttacgc

2641 cgtgggtcga tgtttgatgt tatggagcag caacgatgaa aaagcctgaa ctcaccgcga

2701 cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct

2761 cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc

2821 gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat

2881 cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct

2941 attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc

3001 ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc

3061 agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg

3121 atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca

3181 ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc

3241 ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg


3301 gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg

3361 tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact

3421 tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca

3481 ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg

3541 cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa

3601 tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg

3661 gaaaccgacg ccccagcact cgtccgaggg caaaggaata gccatggatc catggttagc

3721 cctcccacac ataaccagga ggtcagatta tgcagtttaa ggtttacacc tataaaagag

3781 agagccgtta tcgtctgttt gtggatgtac agagtgatat tattgacacg cccgggcgac

3841 ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt

3901 acccggtggt gcatatcggg gatgaaagct ggcgcatgat gaccaccgat atggccagtg

3961 tgccggtctc cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca

4021 aaaacgccat taacctgatg ttctggggaa tataactcga gagatccgaa aaccccaagt

4081 tacggatctt aaggatctcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg

4141 cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac

4201 cttcgggtgg gcctttctgc gtttata

//


Supplementary Note 3: Complete sequence of the pBR322-amp-tetR-tetO-hyg-ccdB plasmid

in Genbank format.

LOCUS pBR322-amp-tetR-tetO-hyg-ccdB 4454 bp DNA circular SYN 27-Jun-2015

DEFINITION pBR322-amp-tetR-tetO-hyg-ccdB

ACCESSION pBR322-amp-tetR-tetO-hyg-ccdB

KEYWORDS

SOURCE

ORGANISM


rep_origin complement(1..683)

/label="pBR322 ori"



/label="amp"

/note="ampicillin resistance gene"


/label="tetR"

/note="tetO repressor gene"

promoter 2435..2516

/label="tetO promoter"

primer_reverse complement(2517..2539)

/label="PCR primer"

CDS 2761..3786

/label="hyg"


CDS 3835..4140

/label="ccdB"


primer 4147..4169

/label="PCR primer"

ORIGIN

1 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg

61 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc

121 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc

181 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc

241 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg

301 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc

361 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga

421 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc

481 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac

541 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg

601 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc


661 acgttaaggg attttggtca tgactagtgc ttggattctc accaataaaa aacgcccggc

721 ggcaaccgag cgttctgaac aaatccagat ggagttctga ggtcattact ggatctatca

781 acaggagtcc aagcgagctc gtaaacttgg tctgacagtt accaatgctt aatcagtgag

841 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg

901 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga

961 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag

1021 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa

1081 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc

1141 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca

1201 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg

1261 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat

1321 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc

1381 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg

1441 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg

1501 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt

1561 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca

1621 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata

1681 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac

1741 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa

1801 gcatgcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc

1861 aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg

1921 taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg

1981 ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa

2041 tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc

2101 atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag

2161 taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc

2221 taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa

2281 agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc

2341 gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt

2401 aatcacttta cttttatcta atctagacat cattaattcc taatttttgt tgacactcta

2461 tcgttgatag agttatttta ccactcccta tcagtgatag agaaaagaat tcaaaagatc

2521 taaagaggag aaaggatctc tcgaggacat aagcctgttc ggttcgtaaa ctgtactgca

2581 agtagcgtat gcgctcacgc aactggtcca gaaccttgac cgaacgcagc ggtggtaacg

2641 gcgcagtggc ggttttcatg gcttgttatg actgtttttt tgtacagtct atgcctcggg

2701 catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg

2761 atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac

2821 agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat

2881 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat

2941 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt

3001 ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg

3061 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat

3121 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga

3181 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat

3241 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag


3301 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc

3361 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg

3421 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct

3481 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg

3541 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac

3601 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga

3661 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc

3721 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag

3781 gaatagccat ggatccatgg ttagccctcc cacacataac caggaggtca gattatgcag

3841 tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga tgtacagagt

3901 gatattattg acacgcccgg gcgacggatg gtgatccccc tggccagtgc acgtctgctg

3961 tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga aagctggcgc

4021 atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga agtggctgat

4081 ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg gggaatataa

4141 ctcgagagat ccgaaaaccc caagttacgg atcttaagga tctccaggca tcaaataaaa

4201 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct

4261 ctctactaga gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacctaggga

4321 gacgtcggcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta

4381 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc

4441 aggaaccgta aaaa

//


Supplementary Note 4: Complete sequence of the pBAC2015 plasmid in Genbank format.

LOCUS pBAC2015 9327 bp DNA circular SYN 27-Jun-2015

DEFINITION pBAC2015

ACCESSION pBAC2015

KEYWORDS

SOURCE

ORGANISM



/label="oriS"


rep_origin 291..909

/label="oriV"



/label="redF"

/note="resolvase"

CDS 1896..2555

/label="cm"

/note="chloramphenicol resistance gene"

misc_feature 2804..2837

/label="loxP511"

/note="mutant loxP site"

promoter 2985..3007

/label="T7 promoter"


/label="EcoRI site"

promoter complement(3177..3194)

/label="Sp6 promoter"

CDS 3293..4711

/label="sacB"

/note="levansucrase encodiing gene"


/label="sopC"

/note="sopC repeat region"


/label="sopB"

/note="F plasmid partitioning protein B"


/label="sopA"

/note="F plasmid partitioning protein A"



/label="repE"

/note="Plasmid F replication protein"

ORIGIN

1 cttctgaggg caatttgtca cagggttaag ggcaatttgt cacagacagg actgtcattt

61 gagggtgatt tgtcacactg aaagggcaat ttgtcacaac accttctcta gaaccagcat

121 ggataaaggc ctacaaggcg ctctaaaaaa gaagatctaa aaactataaa aaaaataatt

181 ataaaaatat ccccgtggat aagtggataa ccccaaggga agttttttca ggcatcgtgt

241 gtaagcagaa tatataagtg ctgttccctg gtgcttcctc gctcactcga ccgggagggt

301 tcgagaaggg ggggcacccc ccttcggcgt gcgcggtcac gcgcacaggg cgcagccctg

361 gttaaaaaca aggtttataa atattggttt aaaagcaggt taaaagacag gttagcggtg

421 gccgaaaaac gggcggaaac ccttgcaaat gctggatttt ctgcctgtgg acagcccctc

481 aaatgtcaat aggtgcgccc ctcatctgtc agcactctgc ccctcaagtg tcaaggatcg

541 cgcccctcat ctgtcagtag tcgcgcccct caagtgtcaa taccgcaggg cacttatccc

601 caggcttgtc cacatcatct gtgggaaact cgcgtaaaat caggcgtttt cgccgatttg

661 cgaggctggc cagctccacg tcgccggccg aaatcgagcc tgcccctcat ctgtcaacgc

721 cgcgccgggt gagtcggccc ctcaagtgtc aacgtccgcc cctcatctgt cagtgagggc

781 caagttttcc gcgaggtatc cacaacgccg gcggccggcc gcggtgtctc gcacacggct

841 tcgacggcgt ttctggcgcg tttgcagggc catagacggc cgccagccca gcggcgaggg

901 caaccagcca gatccgaaaa ccccaagtta cggatctgag ggcttcgccc tgtcgctcaa

961 ctgcggcgag cactactggc tgtaaaagga cagaccacat catggttctg tgttcattag

1021 gttgttctgt ccattgctga cataatccgc tccacttcaa cgtaacaccg cacgaagatt

1081 tctattgttc ctgaaggcat attcaaatcg ttttcgttac cgcttgcagg catcatgaca

1141 gaacactact tcctataaac gctacacagg ctcctgagat taataatgcg gatctctacg

1201 ataatgggag attttcccga ctgtttcgtt cgcttctcag tggataacag ccagcttctc

1261 tgtttaacag acaaaaacag catatccact cagttccaca tttccatata aaggccaagg

1321 catttattct caggataatt gtttcagcat cgcaaccgca tcagactccg gcatcgcaaa

1381 ctgcacccgg tgccgggcag ccacatccag cgcaaaaacc ttcgtgtaga cttccgttga

1441 actgatggac ttatgtccca tcaggctttg cagaactttc agcggtatac cggcatacag

1501 catgtgcatc gcataggaat ggcggaacgt atgtggtgtg accggaacag agaacgtcac

1561 accgtcagca gcagcggcgg caaccgcctc cccaatccag gtcctgaccg ttctgtccgt

1621 cacttcccag atccgcgctt tctctgtcct tcctgtgcga cggttacgcc gctccatgag

1681 cttatcgcga ataaatacct gtgacggaag atcacttcgc agaataaata aatcctggtg

1741 tccctgttga taccgggaag ccctgggcca acttttggcg aaaatgagac gttgatcggc

1801 acgtaagagg ttccaacttt caccataatg aaataagatc actaccgggc gtattttttg

1861 agttatcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata

1921 ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg

1981 ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa

2041 agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg

2101 ctcatccgga gttccgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc

2161 acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat

2221 accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg

2281 aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc

2341 cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc

2401 ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga


2461 ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac

2521 aacagtactg cgatgagtgg cagggcgggg cgtaattttt ttaaggcagt tattggtgcc

2581 cttaaacgcc tggttgctac gcctgaataa gtgataataa gcggatgaat ggcagaaatt

2641 cgatgataag ctgtcaaaca tgagaattgg tcgacggcgc gccaaagctt gcatgcctgc

2701 agccgcgtaa cctggcaaaa tcggttacgg ttgagtaata aatggatgcc ctgcgtaagc

2761 ggggcacatt tcattacctc tttctccgca cccgacatag ataataactt cgtatagtat

2821 acattatacg aagttatcta gtagacttaa ttaaggatcg atccggcgcg ccaatagtca

2881 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag

2941 cttgacattg taggactata ttgctctaat aaatttgcgg ccgctaatac gactcactat

3001 agggagaaaa atatacccgt tatctttcaa gttgcctctt tgttggctgc actcactcac

3061 cccggtcaca tcgttatcta tgctcgggaa ttccggtatc ctctctattt atatgatcaa

3121 gataaatttc cttttgctgt tgatttagtg gaataacaag cggttcaact ttattctata

3181 gtgtcaccta aatgtcgacg gccaggcggc cgccaggcct acccactagt caattcggga

3241 ggatcgaaac ggcagatcgc aaaaaacagt acatacagaa ggagacatga acatgaacat

3301 caaaaaaatt gtaaaacaag ccacagttct gacttttacg actgcacttc tggcaggagg

3361 agcgactcaa gccttcgcga aagaaaataa ccaaaaagca tacaaagaaa cgtacggcgt

3421 ctctcatatt acacgccatg atatgctgca gatccctaaa cagcagcaaa acgaaaaata

3481 ccaagtgcct caattcgatc aatcaacgat taaaaatatt gagtctgcaa aaggacttga

3541 tgtgtgggac agctggccgc tgcaaaacgc tgacggaaca gtagctgaat acaacggcta

3601 tcacgttgtg tttgctcttg cgggaagccc gaaagacgct gatgacacat caatctacat

3661 gttttatcaa aaggtcggcg acaactcaat cgacagctgg aaaaacgcgg gccgtgtctt

3721 taaagacagc gataagttcg acgccaacga tccgatcctg aaagatcaga cgcaagaatg

3781 gtccggttct gcaaccttta catctgacgg aaaaatccgt ttattctaca ctgactattc

3841 cggtaaacat tacggcaaac aaagcctgac aacagcgcag gtaaatgtgt caaaatctga

3901 tgacacactc aaaatcaacg gagtggaaga tcacaaaacg atttttgacg gagacggaaa

3961 aacatatcag aacgttcagc agtttatcga tgaaggcaat tatacatccg gcgacaacca

4021 tacgctgaga gaccctcact acgttgaaga caaaggccat aaataccttg tattcgaagc

4081 caacacggga acagaaaacg gataccaagg cgaagaatct ttatttaaca aagcgtacta

4141 cggcggcggc acgaacttct tccgtaaaga aagccagaag cttcagcaga gcgctaaaaa

4201 acgcgatgct gagttagcga acggcgccct cggtatcata gagttaaata atgattacac

4261 attgaaaaaa gtaatgaagc cgctgatcac ttcaaacacg gtaactgatg aaatcgagcg

4321 cgcgaatgtt ttcaaaatga acggcaaatg gtacttgttc actgattcac gcggttcaaa

4381 aatgacgatc gatggtatta actcaaacga tatttacatg cttggttatg tatcaaactc

4441 tttaaccggc ccttacaagc cgctgaacaa aacagggctt gtgctgcaaa tgggtcttga

4501 tccaaacgat gtgacattca cttactctca cttcgcagtg ccgcaagcca aaggcaacaa

4561 tgtggttatc acaagctaca tgacaaacag aggcttcttc gaggataaaa aggcaacatt

4621 tgcgccaagc ttcttaatga acatcaaagg caataaaaca tccgttgtca aaaacagcat

4681 cctggagcaa ggacagctga cagtcaacta ataacagcaa aaagaaaatg ccgatacttc

4741 attggcattt tcttttattt ctcaacaaga tggtgaattg actagtgggt agatccacag

4801 gacgggtgtg gtcgccatga tcgcgtagtc gatagtggct ccaagtagcg aagcgagcag

4861 gactgggcgg cggccaaagc ggtcggacag tgctccgaga acgggtgcgc atagaaattg

4921 catcaacgca tatagcgcta ggtccctcga ctatagggtc accgtcgaca gcgacacact

4981 tgcatcggat gcagcccggt taacgtgccg gcacggcctg ggtaaccagg tattttgtcc

5041 acataaccgt gcgcaaaatg ttgtggataa gcaggacaca gcagcaatcc acagcaggca


5101 tacaaccgca caccgaggtt actccgttct acaggttacg acgacatgtc aatacttgcc

5161 cttgacaggc attgatggaa tcgtagtctc acgctgatag tctgatcgac aatacaagtg

5221 ggaccgtggt cccagaccga taatcagacc gacaacacga gtgggatcgt ggtcccagac

5281 taataatcag accgacgata cgagtgggac cgtggtccca gactaataat cagaccgacg

5341 atacgagtgg gaccgtggtt ccagactaat aatcagaccg acgatacgag tgggaccgtg

5401 gtcccagact aataatcaga ccgacgatac gagtgggacc atggtcccag actaataatc

5461 agaccgacga tacgagtggg accgtggtcc cagtctgatt atcagaccga cgatacgagt

5521 gggaccgtgg tcccagacta ataatcagac cgacgatacg agtgggaccg tggtcccaga

5581 ctaataatca gaccgacgat acgagtggga ccgtggtccc agtctgatta tcagaccgac

5641 gatacaagtg gaacagtggg cccagagaga atattcaggc cagttatgct ttctggcctg

5701 taacaaagga cattaagtaa agacagataa acgtagacta aaacgtggtc gcatcagggt

5761 gctggctttt caagttcctt aagaatggcc tcaattttct ctatacactc agttggaaca

5821 cgagacctgt ccaggttaag caccatttta tcgcccttat acaatactgt cgctccagga

5881 gcaaactgat gtcgtgagct taaactagtt cttgatgcag atgacgtttt aagcacagaa

5941 gttaaaagag tgataacttc ttcagcttca aatatcaccc cagctttttt ctgctcatga

6001 aggttagatg cctgctgctt aagtaattcc tctttatctg taaaggcttt ttgaagtgca

6061 tcacctgacc gggcagatag ttcaccgggg tgagaaaaaa gagcaacaac tgatttaggc

6121 aatttggcgg tgttgataca gcgggtaata atcttacgtg aaatattttc cgcatcagcc

6181 agcgcagaaa tatttccagc aaattcattc tgcaatcggc ttgcataacg ctgaccacgt

6241 tcataagcac ttgttgggcg ataatcgtta cccaatctgg ataatgcagc catctgctca

6301 tcatccagct cgccaaccag aacacgataa tcactttcgg taagtgcagc agctttacga

6361 cggcgactcc catcggcaat ttctatgaca ccagatactc ttcgaccgaa cgccggtgtc

6421 tgttgaccag tcagtagaaa agaagggatg agatcatcca gtgcgtcctc agtaagcagc

6481 tcctggtcac gttcattacc tgaccatacc cgagaggtct tctcaacact atcaccccgg

6541 agcacttcaa gagtaaactt cacatcccga ccacatacag gcaaagtaat ggcattaccg

6601 cgagccatta ctcctacgcg cgcaattaac gaatccacca tcggggcagc tggtgtcgat

6661 aacgaagtat cttcaaccgg ttgagtattg agcgtatgtt ttggaataac aggcgcacgc

6721 ttcattatct aatctcccag cgtggtttaa tcagacgatc gaaaatttca ttgcagacag

6781 gttcccaaat agaaagagca tttctccagg caccagttga agagcgttga tcaatggcct

6841 gttcaaaaac agttctcatc cggatctgac ctttaccaac ttcatccgtt tcacgtacaa

6901 cattttttag aaccatgctt ccccaggcat cccgaatttg ctcctccatc cacggggact

6961 gagagccatt actattgctg tatttggtaa gcaaaatacg tacatcaggc tcgaaccctt

7021 taagatcaac gttcttgagc agatcacgaa gcatatcgaa aaactgcagt gcggaggtgt

7081 agtcaaacaa ctcagcaggc gtgggaacaa tcagcacatc agcagcacat acgacattaa

7141 tcgtgccgat acccaggtta ggcgcgctgt caataactat gacatcatag tcatgagcaa

7201 cagtttcaat ggccagtcgg agcatcaggt gtggatcggt gggcagttta ccttcatcaa

7261 atttgcccat taactcagtt tcaatacggt gcagagccag acaggaagga ataatgtcaa

7321 gccccggcca gcaagtgggc tttattgcat aagtgacatc gtccttttcc ccaagataga

7381 aaggcaggag agtgtcttct gcatgaatat gaagatctgg tacccatccg tgatacattg

7441 aggctgttcc ctgggggtcg ttaccttcca cgagcaaaac acgtagcccc ttcagagcca

7501 gatcctgagc aagatgaaca gaaactgagg ttttgtaaac gccaccttta tgggcagcaa

7561 ccccgatcac cggtggaaat acgtcttcag cacgtcgcaa tcgcgtacca aacacatcac

7621 gcatatgatt aatttgttca attgtataac caacacgttg ctcaacccgt cctcgaattt

7681 ccatatccgg gtgcggtagt cgccctgctt tctcggcatc tctgatagcc tgagaagaaa


7741 ccccaactaa atccgctgct tcacctattc tccagcgccg ggttattttc ctcgcttccg

7801 ggctgtcatc attaaactgt gcaatggcga tagccttcgt catttcatga ccagcgttta

7861 tgcactggtt aagtgtttcc atgagtttca ttctgaacat cctttaatca ttgctttgcg

7921 tttttttatt aaatcttgca atttactgca aagcaacaac aaaatcgcaa agtcatcaaa

7981 aaaccgcaaa gttgtttaaa ataagagcaa cactacaaaa ggagataaga agagcacata

8041 cctcagtcac ttattatcac tagcgctcgc cgcagccgtg taaccgagca tagcgagcga

8101 actggcgagg aagcaaagaa gaactgttct gtcagatagc tcttacgctc agcgcaagaa

8161 gaaatatcca ccgtgggaaa aactccaggt agaggtacac acgcggatag ccaattcaga

8221 gtaataaact gtgataatca accctcatca atgatgacga actaaccccc gatatcaggt

8281 cacatgacga agggaaagag aaggaaatca actgtgacaa actgccctca aatttggctt

8341 ccttaaaaat tacagttcaa aaagtatgag aaaatccatg caggctgaag gaaacagcaa

8401 aactgtgaca aattaccctc agtaggtcag aacaaatgtg acgaaccacc ctcaaatctg

8461 tgacagataa ccctcagact atcctgtcgt catggaagtg atatcgcgga aggaaaatac

8521 gatatgagtc gtctggcggc ctttcttttt ctcaatgtat gagaggcgca ttggagttct

8581 gctgttgatc tcattaacac agacctgcag gaagcggcgg cggaagtcag gcatacgctg

8641 gtaactttga ggcagctggt aacgctctat gatccagtcg attttcagag agacgatgcc

8701 tgagccatcc ggcttacgat actgacacag ggattcgtat aaacgcatgg catacggatt

8761 ggtgatttct tttgtttcac taagccgaaa ctgcgtaaac cggttctgta acccgataaa

8821 gaagggaatg agatatgggt tgatatgtac actgtaaagc cctctggatg gactgtgcgc

8881 acgtttgata aaccaaggaa aagattcata gcctttttca tcgccggcat cctcttcagg

8941 gcgataaaaa accacttcct tccccgcgaa actcttcaat gcctgccgta tatccttact

9001 ggcttccgca gaggtcaatc cgaatatttc agcatattta gcaacatgga tctcgcagat

9061 accgtcatgt tcctgtaggg tgccatcaga ttttctgatc tggtcaacga acagatacag

9121 catacgtttt tgatcccggg agagactata tgccgcctca gtgaggtcgt ttgactggac

9181 gattcgcggg ctatttttac gtttcttgtg attgataacc gctgtttccg ccatgacaga

9241 tccatgtgaa gtgtgacaag tttttagatt gtcacactaa ataaaaaaga gtcaataagc

9301 agggataact ttgtgaaaaa acagctt

//


Supplementary Note 5: Complete sequence of the pR6K-TnpA-oriT-kan plasmid in Genbank

format.

LOCUS pR6K-TnpA-oriT-kan 3457 bp DNA circular SYN 27-Jun-2015

DEFINITION pR6K-TnpA-oriT-kan

ACCESSION pR6K-TnpA-oriT-kan

KEYWORDS

SOURCE

ORGANISM



/label="R6K ori"

/note="R6K gamma origin of replication"


/label="standard homology arm"

/note="homology sequence to pBR322 and p15A vector"



/note="homology sequence to pBeloBAC11"



/note="homology sequence to pBAC2015 and pBACe3.6"


/label="kan"

/note="kanamycin resistance gene"

misc_feature complement(1689..1715)

/label="IR"

/note="inverted repeat"

CDS 1889..2935

/label="tnpA"

/note="MycoMar transposase encoding gene"

rep_origin 3016..3260

/label="oriT"

/note="RP4 origin of single-stranded DNA transfer"


/label="IR"




/note="homology sequence to pBAC2015, pBACe3.6 and pBeloBAC11"





ORIGIN

1 agttcaacct gttgatagta cgtactaagc tctcatgttt cacgtactaa gctctcatgt

61 ttaacgtact aagctctcat gtttaacgaa ctaaaccctc atggctaacg tactaagctc

121 tcatggctaa cgtactaagc tctcatgttt cacgtactaa gctctcatgt ttgaacaata

181 aaattaatat aaatcagcaa cttaaatagc ctctaaggtt ttaagtttta taagaaaaaa

241 aagaatatat aaggctttta aagcttttaa ggtttaacgg ttgtggacaa caagccaggg

301 atgtaacgca ctgagaagcc cttagagcct ctcaaagcaa ttttgagtga cacaggaaca

361 cttaacggct gacatgggaa ttgctagcat taatactagt gcttggattc tcaccaataa

421 aaaacgcccg gcggcaaccg agcggcatgc tacgtaaatt cactggccgt cgttttacaa

481 cgtcgtgact gggaaactcg agcagatcgc aaaaaacagt acatacagaa ggagacatga

541 actcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg

601 ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca

661 cgggtagcca acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg

721 aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc

781 acgacgagat cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc

841 gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga

901 gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca

961 agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg

1021 tgagatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct

1081 tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc

1141 cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga

1201 accgggcgcc cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt

1261 tgtgcccagt catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat

1321 ccatcttgtt caatcatgcg aaacgatcct catcctgtct cttgatcaga tcttgatccc

1381 ctgcgccatc agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca

1441 accttaccag agggcgcccc agctggcaat tccggttcgc ttgctgtcca taaaaccgcc

1501 cagtctagct atcgccatgt aagcccactg caagctacct gctttctctt tgcgcttgcg

1561 ttttcccttg tccagatagc ccagtagctg acattcatcc ggggtcagca ccgtttctgc

1621 ggactggctt tctacgtgtt ccgcttcctt tagcagccct tgcgccctga gtgcttgcgg

1681 cagcgtgaag accggggact tatcagccaa cctgtactag tacaacttat atcgtatccc

1741 agcccgccta atgagcgggc ttttttttga acaaagtata catctgcaga attccagcac

1801 actggcggcc gttactagac cgtccagtct ggcaggccgg aaacatcggt cagcagatag

1861 gctttaccag taagaaggag agtatactat ggaaaaaaag gaatttcgtg ttttgataaa

1921 atactgtttt ctgaagggaa aaaatacggt ggaagcaaaa acttggcttg ataatgagtt

1981 tccggactct gccccaggga aatcaacaat aattgattgg tatgcaaaat tcaagcgtgg

2041 tgaaatgagc acggaggacg gtgaacgcag tggacgcccg aaagaggtgg ttaccgacga

2101 aaacatcaaa aaaatccaca aaatgatttt gaatgaccgt aaaatgaagt tgatcgagat

2161 agcagaggcc ttaaagatat caaaggaacg tgttggtcat atcattcatc aatatttgga

2221 tatgcggaag ctctgtgcaa aatgggtgcc gcgcgagctc acatttgacc aaaaacaaca

2281 acgtgttgat gattctgagc ggtgtttgca gctgttaact cgtaatacac ccgagttttt

2341 ccgtcgatat gtgacaatgg atgaaacatg gctccatcac tacactcctg agtccaatcg

2401 acagtcggct gagtggacag cgaccggtga accgtctccg aagcgtggaa agactcaaaa

2461 gtccgctggc aaagtaatgg cctctgtttt ttgggatgcg catggaataa tttttatcga

2521 ttatcttgag aagggaaaaa ccatcaacag tgactattat atggcgttat tggagcgttt


2581 gaaggtcgaa atcgcggcaa aacggcccca catgaagaag aaaaaagtgt tgttccacca

2641 agacaacgca ccgtgccaca agtcattgag aacgatggca aaaattcatg aattgggctt

2701 cgaattgctt ccccacccac cgtattctcc agatctggcc cccagcgact ttttcttgtt

2761 ctcagacctc aaaaggatgc tcgcagggaa aaaatttggc tgcaatgaag aggtgatcgc

2821 cgaaactgag gcctattttg aggcaaaacc gaaggagtac taccaaaatg gtatcaaaaa

2881 attggaaggt cgttataatc gttgtatcgc tcttgaaggg aactatgttg aataagcttg

2941 ttatgactgt ttttttgtac agtctatgcc tcgggcccag cactcgtccg agggcaaagg

3001 aataggatcc gcttacggcc agcctcgcag agcaggattc ccgttgagca ccgccaggtg

3061 cgaataaggg acagtgaaga aggaacaccc gctcgcgggt gggcctactt cacctatcct

3121 gcccgggtcg acgccgttgg atacaccaag gaaagtctac acgaaccctt tggcaaaatc

3181 ctgtatatcg tgcgaaaaag gatggatata ccgaaaaaat cgctataatg accccgaagc

3241 agggttatgc agcggaaaag gggagtaaca ggttggctga taagtccccg gtctctgtcg

3301 acagcgacac acttgcatcg gatgcagccc ggttaacgtg ccggcatacg tagcatgctt

3361 aagacccact ttcacattta agttgttttt ctaatccgca tatgatcaat taatgctagc

3421 ccacgatgcg tccggcgtag aggatctgaa gatcagc

//


Supplementary Note 6: Complete sequence of the pR6K-TnpA-oriT-apra plasmid in Genbank

format.

LOCUS pR6K-TnpA-oriT-apra 3208 bp DNA circular SYN 27-Jun-2015

DEFINITION pR6K-TnpA-oriT-apra

ACCESSION pR6K-TnpA-oriT-apra

KEYWORDS

SOURCE

ORGANISM



/label="R6K"












/label="apra"

/note="apramycin resistance gene"


/label="IR"


CDS 1640..2686

/label="tnpA"



/label="oriT"



/label="IR"









ORIGIN









481 cgtcgtgact gggaaaccat ggcagatcgc aaaaaacagt acatacagaa ggagacatga

541 actcagccaa tcgactggcg agcggcatcg cattcttcgc atcccgcctc tggcggatgc

601 aggaagatca acggatctcg gcccagttga cccagggctg tcgccacaat gtcgcgggag

661 cggatcaacc gagcaaaggc atgaccgact ggaccttcct tctgaaggct cttctccttg

721 agccacctgt ccgccaaggc aaagcgctca cagcagtggt cattctcgag ataatcgacg

781 cgtaccaact tgccatcctg aagaatggtg cagtgtctcg gcaccccata gggaaccttt

841 gccatcaact cggcaagatg cagcgtcgtg ttggcatcgt gtcccacgcc gaggagaagt

901 acctgcccat cgagttcatg gacacgggcg accgggcttg caggcgagtg aggtggcagg

961 ggcaatggat cagagatgat ctgctctgcc tgtggccccg ctgccgcaaa ggcaaatgga

1021 tgggcgctgc gctttacatt tggcaggcgc cagaatgtgt cagagacaac tccaaggtcc

1081 ggtgtaacgg gcgacgtggc aggatcgaac ggctcgtcgt ccagacctga ccacgagggc

1141 atgacgagcg tccctcccgg acccagcgca gcacgcaggg cctcgatcag tccaagtggc

1201 ccatcttcga ggggccggac gctacggaag gagctgtgga ccagcagcac accgccgggg

1261 gtaaccccaa ggttgagaag ctgaccgatg agctcggctt ttcgccattc gtattgcacg

1321 acattgcact ccaccgctga tgacatcagt cgatcatagc acgatcaacg gcactgttgc

1381 aaatagtcgg tggtgataaa cttatcatcc ccttttgctg atggagctgc acatgaacca

1441 gaccggggac ttatcagcca acctgtacta gtacaactta tatcgtatcc cagcccgcct

1501 aatgagcggg cttttttttg aacaaagtat acatctgcag aattccagca cactggcggc

1561 cgttactaga ccgtccagtc tggcaggccg gaaacatcgg tcagcagata ggctttacca

1621 gtaagaagga gagtatacta tggaaaaaaa ggaatttcgt gttttgataa aatactgttt

1681 tctgaaggga aaaaatacgg tggaagcaaa aacttggctt gataatgagt ttccggactc

1741 tgccccaggg aaatcaacaa taattgattg gtatgcaaaa ttcaagcgtg gtgaaatgag

1801 cacggaggac ggtgaacgca gtggacgccc gaaagaggtg gttaccgacg aaaacatcaa

1861 aaaaatccac aaaatgattt tgaatgaccg taaaatgaag ttgatcgaga tagcagaggc

1921 cttaaagata tcaaaggaac gtgttggtca tatcattcat caatatttgg atatgcggaa

1981 gctctgtgca aaatgggtgc cgcgcgagct cacatttgac caaaaacaac aacgtgttga

2041 tgattctgag cggtgtttgc agctgttaac tcgtaataca cccgagtttt tccgtcgata

2101 tgtgacaatg gatgaaacat ggctccatca ctacactcct gagtccaatc gacagtcggc

2161 tgagtggaca gcgaccggtg aaccgtctcc gaagcgtgga aagactcaaa agtccgctgg

2221 caaagtaatg gcctctgttt tttgggatgc gcatggaata atttttatcg attatcttga

2281 gaagggaaaa accatcaaca gtgactatta tatggcgtta ttggagcgtt tgaaggtcga

2341 aatcgcggca aaacggcccc acatgaagaa gaaaaaagtg ttgttccacc aagacaacgc

2401 accgtgccac aagtcattga gaacgatggc aaaaattcat gaattgggct tcgaattgct

2461 tccccaccca ccgtattctc cagatctggc ccccagcgac tttttcttgt tctcagacct

2521 caaaaggatg ctcgcaggga aaaaatttgg ctgcaatgaa gaggtgatcg ccgaaactga


2581 ggcctatttt gaggcaaaac cgaaggagta ctaccaaaat ggtatcaaaa aattggaagg

2641 tcgttataat cgttgtatcg ctcttgaagg gaactatgtt gaataagctt gttatgactg

2701 tttttttgta cagtctatgc ctcgggccca gcactcgtcc gagggcaaag gaataggatc

2761 cgcttacggc cagcctcgca gagcaggatt cccgttgagc accgccaggt gcgaataagg

2821 gacagtgaag aaggaacacc cgctcgcggg tgggcctact tcacctatcc tgcccgggtc

2881 gacgccgttg gatacaccaa ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc

2941 gtgcgaaaaa ggatggatat accgaaaaaa tcgctataat gaccccgaag cagggttatg

3001 cagcggaaaa ggggagtaac aggttggctg ataagtcccc ggtctctgtc gacagcgaca

3061 cacttgcatc ggatgcagcc cggttaacgt gccggcatac gtagcatgct taagacccac

3121 tttcacattt aagttgtttt tctaatccgc atatgatcaa ttaatgctag cccacgatgc

3181 gtccggcgta gaggatctga agatcagc

//


Supplementary Note 7: Complete sequence of the pR6K-TnpA-oriT-genta plasmid in

Genbank format.

LOCUS pR6K-TnpA-oriT-genta 3078 bp DNA circular SYN 27-Jun-2015

DEFINITION pR6K-TnpA-oriT-genta

ACCESSION pR6K-TnpA-oriT-genta

KEYWORDS

SOURCE

ORGANISM



/label="R6K"












/label="genta"

/note="gentamicin resistance gene"


/label="IR"


CDS 1510..2556

/label="tnpA"



/label="oriT"



/label="IR"









ORIGIN









481 cgtcgtgact gggaaactcg agcagatcgc aaaaaacagt acatacagaa ggagacatga

541 acttaggtgg cggtacttgg gtcgatatca aagtgcatca cttcttcccg tatgcccaac

601 tttgtataga gagccactgc gggatcgtca ccgtaatctg cttgcacgta gatcacataa

661 gcaccaagcg cgttggcctc atgcttgagg agattgatga gcgcggtggc aatgccctgc

721 ctccggtgct cgccggagac tgcgagatca tagatataga tctcactacg cggctgctca

781 aacctgggca gaacgtaagc cgcgagagcg ccaacaaccg cttcttggtc gaaggcagca

841 agcgcgatga atgtcttact acggagcaag ttcccgaggt aatcggagtc cggctgatgt

901 tgggagtagg tggctacgtc tccgaactca cgaccgaaaa gatcaagagc agcccgcatg

961 gatttgactt ggtcagggcc gagcctacat gtgcgaatga tgcccatact tgagccacct

1021 aactttgttt tagggcgact gccctgctgc gtaacatcgt tgctgctgcg taacatcgtt

1081 gctgctccat aacatcaaac atcgacccac ggcgtaacgc gcttgctgct tggatgcccg

1141 aggcatagac tgtacaaaaa aacagtcata acaagccatg aaaaccgcca ctgcgccgtt

1201 accaccgctg cgttcggtca aggttctgga ccagttgcgt gagcgcatac gctacttgca

1261 ttacagttta cgaaccgaac aggcttatgt caactgggtt cgtgccttca gaccggggac

1321 ttatcagcca acctgtacta gtacaactta tatcgtatcc cagcccgcct aatgagcggg

1381 cttttttttg aacaaagtat acatctgcag aattccagca cactggcggc cgttactaga

1441 ccgtccagtc tggcaggccg gaaacatcgg tcagcagata ggctttacca gtaagaagga

1501 gagtatacta tggaaaaaaa ggaatttcgt gttttgataa aatactgttt tctgaaggga

1561 aaaaatacgg tggaagcaaa aacttggctt gataatgagt ttccggactc tgccccaggg

1621 aaatcaacaa taattgattg gtatgcaaaa ttcaagcgtg gtgaaatgag cacggaggac

1681 ggtgaacgca gtggacgccc gaaagaggtg gttaccgacg aaaacatcaa aaaaatccac

1741 aaaatgattt tgaatgaccg taaaatgaag ttgatcgaga tagcagaggc cttaaagata

1801 tcaaaggaac gtgttggtca tatcattcat caatatttgg atatgcggaa gctctgtgca

1861 aaatgggtgc cgcgcgagct cacatttgac caaaaacaac aacgtgttga tgattctgag

1921 cggtgtttgc agctgttaac tcgtaataca cccgagtttt tccgtcgata tgtgacaatg

1981 gatgaaacat ggctccatca ctacactcct gagtccaatc gacagtcggc tgagtggaca

2041 gcgaccggtg aaccgtctcc gaagcgtgga aagactcaaa agtccgctgg caaagtaatg

2101 gcctctgttt tttgggatgc gcatggaata atttttatcg attatcttga gaagggaaaa

2161 accatcaaca gtgactatta tatggcgtta ttggagcgtt tgaaggtcga aatcgcggca

2221 aaacggcccc acatgaagaa gaaaaaagtg ttgttccacc aagacaacgc accgtgccac

2281 aagtcattga gaacgatggc aaaaattcat gaattgggct tcgaattgct tccccaccca

2341 ccgtattctc cagatctggc ccccagcgac tttttcttgt tctcagacct caaaaggatg

2401 ctcgcaggga aaaaatttgg ctgcaatgaa gaggtgatcg ccgaaactga ggcctatttt

2461 gaggcaaaac cgaaggagta ctaccaaaat ggtatcaaaa aattggaagg tcgttataat

2521 cgttgtatcg ctcttgaagg gaactatgtt gaataagctt gttatgactg tttttttgta


2581 cagtctatgc ctcgggccca gcactcgtcc gagggcaaag gaataggatc cgcttacggc

2641 cagcctcgca gagcaggatt cccgttgagc accgccaggt gcgaataagg gacagtgaag

2701 aaggaacacc cgctcgcggg tgggcctact tcacctatcc tgcccgggtc gacgccgttg

2761 gatacaccaa ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc gtgcgaaaaa

2821 ggatggatat accgaaaaaa tcgctataat gaccccgaag cagggttatg cagcggaaaa

2881 ggggagtaac aggttggctg ataagtcccc ggtctctgtc gacagcgaca cacttgcatc

2941 ggatgcagcc cggttaacgt gccggcatac gtagcatgct taagacccac tttcacattt

3001 aagttgtttt tctaatccgc atatgatcaa ttaatgctag cccacgatgc gtccggcgta

3061 gaggatctga agatcagc

//


Supplementary Note 8: Complete sequence of the pR6K-phiC31-oriT-apra plasmid in

Genbank format.

LOCUS pR6K-phiC31-oriT-apra 4796 bp DNA circular SYN 27-Jun-2015

DEFINITION pR6K-phiC31-oriT-apra

ACCESSION pR6K-phiC31-oriT-apra

KEYWORDS

SOURCE

ORGANISM



/label="R6K"

/note="R6K gamma origin"










CDS 669..1445

/label="apra"

/note="apramycin resistance gene"


/label="oriT"



/label="phiC31 attP site"

CDS 2794..4635

/label="int"

/note="phiC31 integrase"







ORIGIN










481 cgtcgtgact gggaaaggat ccgatatcca gatcgcaaaa aacagtacat acagaaggag

541 acatgaacgg ttcatgtgca gctccatcag caaaagggga tgataagttt atcaccaccg

601 actatttgca acagtgccgt tgatcgtgct atgatcgact gatgtcatca gcggtggagt

661 gcaatgtcgt gcaatacgaa tggcgaaaag ccgagctcat cggtcagctt ctcaaccttg

721 gggttacccc cggcggtgtg ctgctggtcc acagctcctt ccgtagcgtc cggcccctcg

781 aagatgggcc acttggactg atcgaggccc tgcgtgctgc gctgggtccg ggagggacgc

841 tcgtcatgcc ctcgtggtca ggtctggacg acgagccgtt cgatcctgcc acgtcgcccg

901 ttacaccgga ccttggagtt gtctctgaca cattctggcg cctgccaaat gtaaagcgca

961 gcgcccatcc atttgccttt gcggcagcgg ggccacaggc agagcagatc atctctgatc

1021 cattgcccct gccacctcac tcgcctgcaa gcccggtcgc ccgtgtccat gaactcgatg

1081 ggcaggtact tctcctcggc gtgggacacg atgccaacac gacgctgcat cttgccgagt

1141 tgatggcaaa ggttccctat ggggtgccga gacactgcac cattcttcag gatggcaagt

1201 tggtacgcgt cgattatctc gagaatgacc actgctgtga gcgctttgcc ttggcggaca

1261 ggtggctcaa ggagaagagc cttcagaagg aaggtccagt cggtcatgcc tttgctcggt

1321 tgatccgctc ccgcgacatt gtggcgacag ccctgggtca actgggccga gatccgttga

1381 tcttcctgca tccgccagag gcgggatgcg aagaatgcga tgccgctcgc cagtcgattg

1441 gctgagctca tgagcggaga acgagatgac gttggagggg caaggtcgcg ctgattgctg

1501 gggcaacacg tggagcggat cggggattgt ctttcttcag ctcgctgatg atatgctgac

1561 gctcaatgcc gtttggcctc cgactaacga aaatcccgca tttggacggc tgatccgatt

1621 ggcacggcgg acggcgaatg gcggagcaga cgctcgtccg ggggcaatga gatatgaaaa

1681 agcctgaact caccgcgacg tatcgggccc tggccagcta gctagagtcg acctgcaggt

1741 ccccggggat cggtcttgcc ttgctcgtcg gtgatgtact tcaccagctc cgcgaagtcg

1801 ctcttcttga tggagcgcat ggggacgtgc ttggcaatca cgcgcacccc ccggccgttt

1861 tagcggctaa aaaagtcatg gctctgccct cgggcggacc acgcccatca tgaccttgcc

1921 aagctcgtcc tgcttctctt cgatcttcgc cagcagggcg aggatcgtgg catcaccgaa

1981 ccgcgccgtg cgcgggtcgt cggtgagcca gagtttcagc aggccgccca ggcggcccag

2041 gtcgccattg atgcgggcca gctcgcggac gtgctcatag tccacgacgc ccgtgatttt

2101 gtagccctgg ccgacggcca gcaggtaggc cgacaggctc atgccggccg ccgccgcctt

2161 ttcctcaatc gctcttcgtt cgtctggaag gcagtacacc ttgataggtg ggctgccctt

2221 cctggttggc ttggtttcat cagccatccg cttgccctca tctgttacgc cggcggtagc

2281 cggccagcct cgcagagcag gattcccgtt gagcaccgcc aggtgcgaat aagggacagt

2341 gaagaaggaa cacccgctcg cgggtgggcc tacttcacct atcctgcccg gctgacgccg

2401 ttggatacac caaggaaagt ctacacgaac cctttggcaa aatcctgtat atcgtgcgaa

2461 aaaggatgga tataccgaaa aaatcgctat aatgaccccg aagcagggtt atgcagcgga

2521 aaagatccgt cgacctgcag gcatgcaagc tctagcgatt ccagacgtcc cgaaggcgtg

2581 gcgcggcttc cccgtgccgg agcaatcgcc ctgggtgggt tacacgacgc ccctctatgg

2641 cccgtactga cggacacacc gaagccccgg cggcaaccct cagcggatgc cccggggctt

2701 cacgttttcc caggtcagaa gcggttttcg ggagtagtgc cccaactggg gtaacctttg

2761 agttctctca gttgggggcg tagggtcgcc gacatgacac aaggggttgt gaccggggtg


2821 gacacgtacg cgggtgctta cgaccgtcag tcgcgcgagc gcgagaattc gagcgcagca

2881 agcccagcga cacagcgtag cgccaacgaa gacaaggcgg ccgaccttca gcgcgaagtc

2941 gagcgcgacg ggggccggtt caggttcgtc gggcatttca gcgaagcgcc gggcacgtcg

3001 gcgttcggga cggcggagcg cccggagttc gaacgcatcc tgaacgaatg ccgcgccggg

3061 cggctcaaca tgatcattgt ctatgacgtg tcgcgcttct cgcgcctgaa ggtcatggac

3121 gcgattccga ttgtctcgga attgctcgcc ctgggcgtga cgattgtttc cactcaggaa

3181 ggcgtcttcc ggcagggaaa cgtcatggac ctgattcacc tgattatgcg gctcgacgcg

3241 tcgcacaaag aatcttcgct gaagtcggcg aagattctcg acacgaagaa ccttcagcgc

3301 gaattgggcg ggtacgtcgg cgggaaggcg ccttacggct tcgagcttgt ttcggagacg

3361 aaggagatca cgcgcaacgg ccgaatggtc aatgtcgtca tcaacaagct tgcgcactcg

3421 accactcccc ttaccggacc cttcgagttc gagcccgacg taatccggtg gtggtggcgt

3481 gagatcaaga cgcacaaaca ccttcccttc aagccgggca gtcaagccgc cattcacccg

3541 ggcagcatca cggggctttg taagcgcatg gacgctgacg ccgtgccgac ccggggcgag

3601 acgattggga agaagaccgc ttcaagcgcc tgggacccgg caaccgttat gcgaatcctt

3661 cgggacccgc gtattgcggg cttcgccgct gaggtgatct acaagaagaa gccggacggc

3721 acgccgacca cgaagattga gggttaccgc attcagcgcg acccgatcac gctccggccg

3781 gtcgagcttg attgcggacc gatcatcgag cccgctgagt ggtatgagct tcaggcgtgg

3841 ttggacggca gggggcgcgg caaggggctt tcccgggggc aagccattct gtccgccatg

3901 gacaagctgt actgcgagtg tggcgccgtc atgacttcga agcgcgggga agaatcgatc

3961 aaggactctt accgctgccg tcgccggaag gtggtcgacc cgtccgcacc tgggcagcac

4021 gaaggcacgt gcaacgtcag catggcggca ctcgacaagt tcgttgcgga acgcatcttc

4081 aacaagatca ggcacgccga aggcgacgaa gagacgttgg cgcttctgtg ggaagccgcc

4141 cgacgcttcg gcaagctcac tgaggcgcct gagaagagcg gcgaacgggc gaaccttgtt

4201 gcggagcgcg ccgacgccct gaacgccctt gaagagctgt acgaagaccg cgcggcaggc

4261 gcgtacgacg gacccgttgg caggaagcac ttccggaagc aacaggcagc gctgacgctc

4321 cggcagcaag gggcggaaga gcggcttgcc gaacttgaag ccgccgaagc cccgaagctt

4381 ccccttgacc aatggttccc cgaagacgcc gacgctgacc cgaccggccc taagtcgtgg

4441 tgggggcgcg cgtcagtaga cgacaagcgc gtgttcgtcg ggctcttcgt agacaagatc

4501 gttgtcacga agtcgactac gggcaggggg cagggaacgc ccatcgagaa gcgcgcttcg

4561 atcacgtggg cgaagccgcc gaccgacgac gacgaagacg acgcccagga cggcacggaa

4621 gacgtagcgg cgtaggtcga cagcgacaca cttgcatcgg atgcagcccg gttaacgtgc

4681 cggcatacgt agcatgctta agacccactt tcacatttaa gttgtttttc taatccgcat

4741 atgatcaatt aatgctagcc cacgatgcgt ccggcgtaga ggatctgaag atcagc

//


Supplementary Note 9: Complete sequence of the pBBR1-kan-hyg-ccdB plasmid in Genbank

format.

LOCUS pBBR1-kan-hyg-ccdB 6062 bp DNA circular SYN 27-Jun-2015

DEFINITION pBBR1-kan-hyg-ccdB

ACCESSION pBBR1-kan-hyg-ccdB

KEYWORDS

SOURCE

ORGANISM



/label="pBBR1 ori"

/note="broad-host-range origin of replication"

CDS 904..1902

/label="mob"

/note="required for plasmid mobilization (RP4 mob, oriT)"

CDS 2160..2954

/label="kan"





CDS 3546..4571

/label="hyg"


CDS 4620..4925

/label="ccdB"






/label="rep"

/note="required for plasmid replication"

ORIGIN

1 gcgtaaaagt ggcagtcaat tacagatttt ctttaaccta cgcaatgagc tattgcgggg

61 ggtgccgcaa tgagctgttg cgtacccccc ttttttaagt tgttgatttt taagtctttc

121 gcatttcgcc ctatatctag ttctttggtg cccaaagaag ggcacccctg cggggttccc

181 ccacgccttc ggcgcggctc cccctccggc aaaaagtggc ccctccgggg cttgttgatc

241 gactgcgcgg ccttcggcct tgcccaaggt ggcgctgccc ccttggaacc cccgcactcg

301 ccgccgtgag gctcgggggg caggcgggcg ggcttcgcct tcgactgccc ccactcgcat

361 aggcttgggt cgttccaggc gcgtcaaggc caagccgctg cgcggtcgct gcgcgagcct

421 tgacccgcct tccacttggt gtccaaccgg caagcgaagc gcgcaggccg caggccggag


481 gcttttcccc agagaaaatt aaaaaaattg atggggcaag gccgcaggcc gcgcagttgg

541 agccggtggg tatgtggtcg aaggctgggt agccggtggg caatccctgt ggtcaagctc

601 gtgggcaggc gcagcctgtc catcagcttg tccagcaggg ttgtccacgg gccgagcgaa

661 gcgagccagc cggtggccgc tcgcggccat cgtccacata tccacgggct ggcaagggag

721 cgcagcgacc gcgcagggcg aagcccggag agcaagcccg tagggcgccg cagccgccgt

781 aggcggtcac gactttgcga agcaaagtct agtgagtata ctcaagcatt gagtggcccg

841 ccggaggcac cgccttgcgc tgcccccgtc gagccggttg gacaccaaaa gggaggggca

901 ggcatggcgg catacgcgat catgcgatgc aagaagctgg cgaaaatggg caacgtggcg

961 gccagtctca agcacgccta ccgcgagcgc gagacgccca acgctgacgc cagcaggacg

1021 ccagagaacg agcactgggc ggccagcagc accgatgaag cgatgggccg actgcgcgag

1081 ttgctgccag agaagcggcg caaggacgct gtgttggcgg tcgagtacgt catgacggcc

1141 agcccggaat ggtggaagtc ggccagccaa gaacagcagg cggcgttctt cgagaaggcg

1201 cacaagtggc tggcggacaa gtacggggcg gatcgcatcg tgacggccag catccaccgt

1261 gacgaaacca gcccgcacat gaccgcgttc gtggtgccgc tgacgcagga cggcaggctg

1321 tcggccaagg agttcatcgg caacaaagcg cagatgaccc gcgaccagac cacgtttgcg

1381 gccgctgtgg ccgatctagg gctgcaacgg ggcatcgagg gcagcaaggc acgtcacacg

1441 cgcattcagg cgttctacga ggccctggag cggccaccag tgggccacgt caccatcagc

1501 ccgcaagcgg tcgagccacg cgcctatgca ccgcagggat tggccgaaaa gctgggaatc

1561 tcaaagcgcg ttgagacgcc ggaagccgtg gccgaccggc tgacaaaagc ggttcggcag

1621 gggtatgagc ctgccctaca ggccgccgca ggagcgcgtg agatgcgcaa gaaggccgat

1681 caagcccaag agacggcccg agaccttcgg gagcgcctga agcccgttct ggacgccctg

1741 gggccgttga atcgggatat gcaggccaag gccgccgcga tcatcaaggc cgtgggcgaa

1801 aagctgctga cggaacagcg ggaagtccag cgccagaaac aggcccagcg ccagcaggaa

1861 cgcgggcgcg cacatttccc cgaaaagtgc cacctgggat gaatgtcagc tactgggcta

1921 tctggacaag ggaaaacgca agcgcaaaga gaaagcaggt agcttgcagt gggcttacat

1981 ggcgatagct agactgggcg gttttatgga cagcaagcga accggaattg ccagctgggg

2041 cgccctctgg taaggttggg aagccctgca aagtaaactg gatggctttc ttgccgccaa

2101 ggatctgatg gcgcagggga tcaagatctg atcaagagac aggatgagga tcgtttcgca

2161 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg

2221 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag

2281 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc

2341 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc

2401 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg

2461 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc

2521 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca

2581 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag

2641 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg

2701 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg

2761 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca

2821 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc

2881 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg

2941 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct

3001 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt

3061 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc


3121 ccacccccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc

3181 aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac

3241 agtttttatg cattaagacc cactttcaca tttaagttgt ttttctaatc cgcatatgat

3301 caattcaagg ccgtgcactg tacaggatcc gacataagcc tgttcggttc gtaaactgta

3361 ctgcaagtag cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac gcagcggtgg

3421 taacggcgca gtggcggttt tcatggcttg ttatgactgt ttttttgtac agtctatgcc

3481 tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag

3541 caacgatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt

3601 tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct

3661 tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca

3721 aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg

3781 acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca

3841 cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca

3901 tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc

3961 aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg

4021 tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg

4081 atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt

4141 tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg

4201 aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt

4261 tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat

4321 cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg

4381 ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat

4441 ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg

4501 atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg

4561 caaaggaata gccatggatc catggttagc cctcccacac ataaccagga ggtcagatta

4621 tgcagtttaa ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac

4681 agagtgatat tattgacacg cccgggcgac ggatggtgat ccccctggcc agtgcacgtc

4741 tgctgtcaga taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct

4801 ggcgcatgat gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg

4861 ctgatctcag ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa

4921 tataaggatc ctgtacagtg cactgaacgc tctctactag agtcacactg gctcaccttc

4981 gggtgggcct ttctgcgttt atagcttacc ggtttattga ctaccggaag cagtgtgacc

5041 gtgtgcttct caaatgcctg aggccagttt gctcaggctc tccccgtgga ggtaataatt

5101 gacgatatga tcatttattc tgcctcccag agcctgataa aaacggtgaa tccgttagcg

5161 aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg

5221 cggggaggca gacaaggtat agggcggcga ggcggctaca gccgatagtc tggaacagcg

5281 cacttacggg ttgctgcgca acccaagtgc taccggcgcg gcagcgtgac ccgtgtcggc

5341 ggctccaacg gctcgccatc gtccagaaaa cacggctcat cgggcatcgg caggcgctgc

5401 tgcccgcgcc gttcccattc ctccgtttcg gtcaaggctg gcaggtctgg ttccatgccc

5461 ggaatgccgg gctggctggg cggctcctcg ccggggccgg tcggtagttg ctgctcgccc

5521 ggatacaggg tcgggatgcg gcgcaggtcg ccatgcccca acagcgattc gtcctggtcg

5581 tcgtgatcaa ccaccacggc ggcactgaac accgacaggc gcaactggtc gcggggctgg

5641 ccccacgcca cgcggtcatt gaccacgtag gccgacacgg tgccggggcc gttgagcttc

5701 acgacggaga tccagcgctc ggccaccaag tccttgactg cgtattggac cgtccgcaaa


5761 gaacgtccga tgagcttgga aagtgtcttc tggctgacca ccacggcgtt ctggtggccc

5821 atctgcgcca cgaggtgatg cagcagcatt gccgccgtgg gtttcctcgc aataagcccg

5881 gcccacgcct catgcgcttt gcgttccgtt tgcacccagt gaccgggctt gttcttggct

5941 tgaatgccga tttctctgga ctgcgtggcc atgcttatct ccatgcggta gggtgccgca

6001 cggttgcggc accatgcgca atcagctgca acttttcggc agcgcgacaa caattatgcg

6061 tt

//


Supplementary Note 10: Complete sequence of the pBR322-amp-ccdB-rpsL plasmid in

Genbank format.

LOCUS pBR322-amp-ccdB-rpsL 3352 bp DNA circular SYN 27-Jun-2015

DEFINITION pBR322-amp-ccdB-rpsL

ACCESSION pBR322-amp-ccdB-rpsL

KEYWORDS

SOURCE

ORGANISM


rep_origin 1..620

/label="pBR322 ori"

primer_reverse complement(621..640)

/label="PCR primer"

CDS 788..1162

/label="rpsL"

/note="ribosomal protein S12 encoding gene"

CDS 1174..1968

/label="kan"


primer 1975..1994

/label="PCR primer"


/label="ccdB"



/label="amp"

/note="ampicillin resistance gene"

ORIGIN

1 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa

61 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc

121 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt

181 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc

241 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac

301 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca

361 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg

421 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag

481 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt

541 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat

601 ggaaaaacgc cagcaacgcg cattagccct tgccatttgt gatatcggta ccggcctggt

661 gatggcggga tcgttgtata tttcttgaca ccttttcggc atcgccctaa aattcggcgt

721 cctcatattg tgtgaggacg ttttattacg tgtttacgaa gcaaaagcta aaaccaggag

781 ctatttaatg gcaacagtta accagctggt acgcaaacca cgtgctcgca aagttgcgaa


841 aagcaacgtg cctgcgctgg aagcatgccc gcaaaaacgt ggcgtatgta ctcgtgtata

901 tactaccact cctaaaaaac cgaactccgc gctgcgtaaa gtatgccgtg ttcgtctgac

961 taacggtttc gaagtgactt cctacatcgg tggtgaaggt cacaacctgc aggagcactc

1021 cgtgatcctg atccgtggcg gtcgtgttaa agacctcccg ggtgttcgtt accacaccgt

1081 acgtggtgcg cttgactgct ccggcgttaa agaccgtaag caggctcgtt ccaagtatgg

1141 cgtgaagcgt cctaaggctt aaggaggaca atcatgattg aacaagatgg attgcacgca

1201 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc

1261 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc

1321 aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg

1381 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg

1441 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct

1501 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct

1561 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa

1621 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa

1681 ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt gacccatggc

1741 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt

1801 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct

1861 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc

1921 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgaga tatcggtgtg

1981 gtagctcgcg tattttttgt tcaaaaaaaa gccagctcat taggcgggct gggttatatt

2041 ccccagaaca tcaggttaat ggcgtttttg atgtcatttt cgcggtggct gagatcagcc

2101 acttcttccc cgataacgga gaccggcaca ctggccatat cggtggtcat catgcgccag

2161 ctttcatccc cgatatgcac caccgggtaa agttcacggg agactttatc tgacagcaga

2221 cgtgcactgg ccagggggat caccatccgt cgcccgggcg tgtcaataat atcactctgt

2281 acatccacaa acagacgata acggctctct cttttatagg tgtaaacctt aaactgcata

2341 atctgacctc ctggttatgt gtgggagggc taaccatgga tccatggtta ccaatgctta

2401 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc

2461 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg

2521 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga

2581 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt

2641 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt

2701 gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc

2761 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc

2821 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca

2881 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag

2941 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg

3001 tcaacacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa

3061 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa

3121 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga

3181 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga

3241 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg

3301 agcggataca tatttgaatg tatttagaaa aataaacaaa ggatccgaat tc

//


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