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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
Supplementary Figure 2
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.
Nature Protocols: doi:10.1038/nprot.2016.054
Supplementary Figure 3
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.
Nature Protocols: doi:10.1038/nprot.2016.054
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
Nature Protocols: doi:10.1038/nprot.2016.054
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)
Supplementary note 6 This study
pR6K-oriT-TnpA-genta Suicide plasmid to release a conjugation-transposition cassette to modify a cloning vector (PCR free recombineering)
Supplementary note 7 This study
pR6K-oriT-phiC31 Suicide plasmid to release a conjugation-site-specific-integration cassette to modify a cloning vector (PCR free recombineering)
Supplementary note 8 This study
pBBR1-kan-hyg-ccdB Suicide plasmid to release a linear broad-host-range vector for subcloning (PCR free recombineering)
Supplementary note 9 This study
pBR322-amp-ccdB-rpsL PCR template of pBR322-amp-ccdB for pBAC2015 modification to introduce homology sequences for direct cloning
Supplementary note 10 This study
Nature Protocols: doi:10.1038/nprot.2016.054
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
Nature Protocols: doi:10.1038/nprot.2016.054
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.
Nature Protocols: doi:10.1038/nprot.2016.054
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).
Nature Protocols: doi:10.1038/nprot.2016.054
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
Nature Protocols: doi:10.1038/nprot.2016.054
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
Nature Protocols: doi:10.1038/nprot.2016.054
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
Nature Protocols: doi:10.1038/nprot.2016.054
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
Nature Protocols: doi:10.1038/nprot.2016.054
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
//
Nature Protocols: doi:10.1038/nprot.2016.054
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
FEATURES Location/Qualifiers
rep_origin 1..718
/label="p15A ori"
/note="replication origin"
CDS complement(930..1589)
/label="cm"
/note="chloramphenicol resistance gene"
CDS complement(1722..2348)
/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
Nature Protocols: doi:10.1038/nprot.2016.054
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
Nature Protocols: doi:10.1038/nprot.2016.054
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
//
Nature Protocols: doi:10.1038/nprot.2016.054
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
FEATURES Location/Qualifiers
rep_origin complement(1..683)
/label="pBR322 ori"
/note="replication origin"
CDS complement(819..1679)
/label="amp"
/note="ampicillin resistance gene"
CDS complement(1807..2433)
/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"
/note="hygromycin resistance gene"
CDS 3835..4140
/label="ccdB"
/note="CcdB toxic protein"
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
Nature Protocols: doi:10.1038/nprot.2016.054
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
Nature Protocols: doi:10.1038/nprot.2016.054
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
//
Nature Protocols: doi:10.1038/nprot.2016.054
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
FEATURES Location/Qualifiers
rep_origin complement(1..290)
/label="oriS"
/note="replication origin"
rep_origin 291..909
/label="oriV"
/note="replication origin"
CDS complement(1330..1677)
/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"
misc_feature 3088..3093
/label="EcoRI site"
promoter complement(3177..3194)
/label="Sp6 promoter"
CDS 3293..4711
/label="sacB"
/note="levansucrase encodiing gene"
misc_feature 5208..5681
/label="sopC"
/note="sopC repeat region"
CDS complement(5754..6725)
/label="sopB"
/note="F plasmid partitioning protein B"
CDS complement(6725..7900)
/label="sopA"
/note="F plasmid partitioning protein A"
CDS complement(8479..9234)
Nature Protocols: doi:10.1038/nprot.2016.054
/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
Nature Protocols: doi:10.1038/nprot.2016.054
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
Nature Protocols: doi:10.1038/nprot.2016.054
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
Nature Protocols: doi:10.1038/nprot.2016.054
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
//
Nature Protocols: doi:10.1038/nprot.2016.054
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
FEATURES Location/Qualifiers
rep_origin complement(1..377)
/label="R6K ori"
/note="R6K gamma origin of replication"
misc_feature 395..444
/label="standard homology arm"
/note="homology sequence to pBR322 and p15A vector"
misc_feature 457..496
/label="standard homology arm"
/note="homology sequence to pBeloBAC11"
misc_feature 503..542
/label="standard homology arm"
/note="homology sequence to pBAC2015 and pBACe3.6"
CDS complement(543..1337)
/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"
misc_feature 3268..3294
/label="IR"
/note="inverted repeat"
misc_feature 3297..3346
/label="standard homology arm"
/note="homology sequence to pBAC2015, pBACe3.6 and pBeloBAC11"
misc_feature 3359..3408
/label="standard homology arm"
/note="homology sequence to pBR322 and p15A vector"
Nature Protocols: doi:10.1038/nprot.2016.054
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
Nature Protocols: doi:10.1038/nprot.2016.054
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
//
Nature Protocols: doi:10.1038/nprot.2016.054
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
FEATURES Location/Qualifiers
rep_origin complement(1..377)
/label="R6K"
/note="R6K gamma origin of replication"
misc_feature 395..444
/label="standard homology arm"
/note="homology sequence to pBR322 and p15A vector"
misc_feature 457..496
/label="standard homology arm"
/note="homology sequence to pBeloBAC11"
misc_feature 503..542
/label="standard homology arm"
/note="homology sequence to pBAC2015 and pBACe3.6"
CDS complement(543..1319)
/label="apra"
/note="apramycin resistance gene"
misc_feature complement(1440..1466)
/label="IR"
/note="inverted repeat"
CDS 1640..2686
/label="tnpA"
/note="MycoMar transposase encoding gene"
rep_origin 2767..3011
/label="oriT"
/note="RP4 origin of single-stranded DNA transfer"
misc_feature 3019..3045
/label="IR"
/note="inverted repeat"
misc_feature 3110..3159
/label="standard homology arm"
/note="homology sequence to pBR322 and p15A vector"
misc_feature 3048..3097
/label="standard homology arm"
/note="homology sequence to pBAC2015, pBACe3.6 and pBeloBAC11"
Nature Protocols: doi:10.1038/nprot.2016.054
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 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
Nature Protocols: doi:10.1038/nprot.2016.054
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
//
Nature Protocols: doi:10.1038/nprot.2016.054
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
FEATURES Location/Qualifiers
rep_origin complement(1..377)
/label="R6K"
/note="R6K gamma origin of replication"
misc_feature 395..444
/label="standard homology arm"
/note="homology sequence to pBR322 and p15A vector"
misc_feature 457..496
/label="standard homology arm"
/note="homology sequence to pBeloBAC11"
misc_feature 503..542
/label="standard homology arm"
/note="homology sequence to pBAC2015 and pBACe3.6"
CDS complement(543..1076)
/label="genta"
/note="gentamicin resistance gene"
misc_feature complement(1310..1336)
/label="IR"
/note="inverted repeat"
CDS 1510..2556
/label="tnpA"
/note="MycoMar transposase encoding gene"
rep_origin 2637..2881
/label="oriT"
/note="RP4 origin of single-stranded DNA transfer"
misc_feature 2889..2915
/label="IR"
/note="inverted repeat"
misc_feature 2918..2967
/label="standard homology arm"
/note="homology sequence to pBAC2015, pBACe3.6 and pBeloBAC11"
misc_feature 2980..3029
/label="standard homology arm"
/note="homology sequence to pBR322 and p15A vector"
Nature Protocols: doi:10.1038/nprot.2016.054
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 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
Nature Protocols: doi:10.1038/nprot.2016.054
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
//
Nature Protocols: doi:10.1038/nprot.2016.054
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
FEATURES Location/Qualifiers
rep_origin complement(1..377)
/label="R6K"
/note="R6K gamma origin"
misc_feature 395..444
/label="standard homology arm"
/note="homology sequence to pBR322 and p15A vector"
misc_feature 457..496
/label="standard homology arm"
/note="homology sequence to pBeloBAC11"
misc_feature 509..548
/label="standard homology arm"
/note="homology sequence to pBAC2015 and pBACe3.6"
CDS 669..1445
/label="apra"
/note="apramycin resistance gene"
rep_origin 2280..2391
/label="oriT"
/note="RP4 origin of single-stranded DNA transfer"
misc_feature 2739..2777
/label="phiC31 attP site"
CDS 2794..4635
/label="int"
/note="phiC31 integrase"
misc_feature 4636..4685
/label="standard homology arm"
/note="homology sequence to pBAC2015, pBACe3.6 and pBeloBAC11"
misc_feature 4698..4747
/label="standard homology arm"
/note="homology sequence to pBR322 and p15A vector"
ORIGIN
1 agttcaacct gttgatagta cgtactaagc tctcatgttt cacgtactaa gctctcatgt
61 ttaacgtact aagctctcat gtttaacgaa ctaaaccctc atggctaacg tactaagctc
121 tcatggctaa cgtactaagc tctcatgttt cacgtactaa gctctcatgt ttgaacaata
Nature Protocols: doi:10.1038/nprot.2016.054
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 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
Nature Protocols: doi:10.1038/nprot.2016.054
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
//
Nature Protocols: doi:10.1038/nprot.2016.054
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
FEATURES Location/Qualifiers
rep_origin complement(1..818)
/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"
/note="kanamycin resistance gene"
misc_feature 3253..3312
/label="standard homology arm"
/note="homology sequence to pBR322 and p15A vector"
CDS 3546..4571
/label="hyg"
/note="hygromycin resistance gene"
CDS 4620..4925
/label="ccdB"
/note="CcdB toxic protein"
misc_feature 4944..5003
/label="standard homology arm"
/note="homology sequence to pBR322 and p15A vector"
CDS complement(5310..5984)
/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
Nature Protocols: doi:10.1038/nprot.2016.054
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
Nature Protocols: doi:10.1038/nprot.2016.054
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
Nature Protocols: doi:10.1038/nprot.2016.054
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
//
Nature Protocols: doi:10.1038/nprot.2016.054
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
FEATURES Location/Qualifiers
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"
/note="kanamycin resistance gene"
primer 1975..1994
/label="PCR primer"
CDS complement(2034..2339)
/label="ccdB"
/note="CcdB toxic protein"
CDS complement(2388..3248)
/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
Nature Protocols: doi:10.1038/nprot.2016.054
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
//
Nature Protocols: doi:10.1038/nprot.2016.054