LOCUS       40924_33932        6919 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Chromosomal integration vector pOSIP-TO, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6919)
  AUTHORS   St-Pierre F, Cui L, Priest DG, Endy D, Dodd IB, Shearwin KE.
  TITLE     One-step cloning and chromosomal integration of DNA
  JOURNAL   ACS Synth Biol 2 (9), 537-541 (2013)
  PUBMED    24050148
REFERENCE   2  (bases 1 to 6919)
  AUTHORS   St-Pierre F, Cui L, Priest DG, Endy D, Dodd IB, Shearwin KE.
  TITLE     Direct Submission
  JOURNAL   Submitted (12-MAY-2013) Molecular and Biomedical Science, The 
            University of Adelaide, Molecular Life Sciences Building, Adelaide, 
            South Australia 5005, Australia
REFERENCE   3  (bases 1 to 6919)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6919)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol"; date: "2013"; volume: "2"; issue: "9"; pages: "537-541"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (12-MAY-2013) Molecular and Biomedical Science, The University of 
            Adelaide, Molecular Life Sciences Building, Adelaide, South 
            Australia 5005, Australia"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6919
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             142..444
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
     rep_origin      477..1065
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    1125..1145
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1200..1227
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(1276..1307)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription
                     terminator"
     protein_bind    1384..1417
                     /label=FRT (minimal)
                     /note="supports FLP-mediated excision but not integration
                     (Turan and Bode, 2011)"
     CDS             complement(1427..2137)
                     /label=lambda repressor (ts)
                     /note="temperature-sensitive variant of the phage lambda
                     repressor"
     RBS             2226..2234
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     RBS             2246..2268
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2276..3283
                     /label=phage 186 integrase
                     /note="integrase from phage 186"
     protein_bind    3406..3424
                     /label=tet operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             3433..4635
                     /label=TcR
                     /note="tetracycline efflux protein"
     terminator      4697..4791
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      4848..5236
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     misc_feature    5247..5294
                     /note="FRT; Flp recombinase target site"
     protein_bind    5247..5294
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     protein_bind    complement(5307..5521)
                     /label=phage 186 attP
                     /note="attachment site of phage 186"
     terminator      complement(5634..5880)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     terminator      6200..6286
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      6381..6467
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      6562..6648
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      complement(6746..6789)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
ORIGIN
        1 gaattcgagc tcggtacccg gggatcctct agagggctta ctaaaagcca gataacagta
       61 tgcgtatttg cgcgctgatt tttgcggtat aagaatatat actgatatgt atacccgaag
      121 tatgtcaaaa agaggtatgc tatgcagttt aaggtttaca cctataaaag agagagccgt
      181 tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg acggatggtg
      241 atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact ttacccggtg
      301 gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag tgtgccggtc
      361 tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat caaaaacgcc
      421 attaacctga tgttctgggg aatataataa cccgtagaaa agatcaaaag atcttcttga
      481 gatccttttt ttctgcgcgt aatctgctac ttgcaaacaa aaaaaccacc gctaccagcg
      541 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggctttagc
      601 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
      661 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
      721 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
      781 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
      841 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
      901 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
      961 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     1021 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     1081 gcctttttac ggttcctggc cttttgctgg ccttttgctc acattactag tagcggccgc
     1141 tgcaggcatg cctcgagatg catggcgcct aacctaaact gacaggcatc aaattaagca
     1201 gaaggccatc ctgacggatg gcctttttgc gtttcgaaca attgaaaaaa cctcgcgcct
     1261 tacctgttga gtaatagtca aaagcctccg gtcggaggct tttgactttc tgcttactga
     1321 atttcggtgg tgccgttaat taaccggtgg gccctcatga taataatggt ttcttagacg
     1381 tccgaagttc ctattctcta gaaagtatag gaacttccct aggtcagcca aacgtctctt
     1441 caggccactg actagcgata actttcccca caacggaaca actctcattg catgggatca
     1501 ttgggtactg tgggtttagt ggttgtaaaa acacctgacc gctatccctg atcagtttct
     1561 tgaaggtaaa ctcatcaccc ccaagtctgg ctatgcagaa atcacctggc tcaacagcct
     1621 gctcagggtc aacgagaatt aacattccgt caggaaagct tggcttggag cctgttggtg
     1681 cggtcatgga attaccttca acctcaagcc agaatgcaga atcactggct tttttggttg
     1741 tgcttaccca tctctccgca tcacctttgg taaaggttct aagcttaggt gagaacatcc
     1801 ctgcctgaac atgagaaaaa acagggtact catactcact tctaagtgac ggctgcatac
     1861 taaccgcttc atacatctcg tagatttctc tggcgattga agggctaaat tcttcaacgc
     1921 taactttgag aatttttgta agcaatgcgg cgttataagc atttaatgca ttgatgccat
     1981 taaataaagc accaacgcct gactgcccca tccccatctt gtctgcgaca gattcctggg
     2041 ataagccaag ttcatttttc tttttttcat aaattgcttt aaggcgacgt gcgtcctcaa
     2101 gctgctcttg tgttaatggt ttcttttttg tgctcatacg ttaaatctat caccgcaagg
     2161 gataaatatc taacaccgtg cgtgttgact attttacctc tggcggtgat aatggttgca
     2221 tgtactaagg aggttgtata cgcgttttgt ttaactttaa gaaggagata tacatatgac
     2281 cgtccgtaaa aatccggctg gcggttggat ttgtgaactc tacccaaacg gtgcaaaagg
     2341 caaacgtatc agaaagaaat tcgctactaa aggcgaggct ctggcgtttg agcagtacac
     2401 cgttcaaaac ccgtggcagg aagaaaagga agacaggcgc acgttaaaag agctggttga
     2461 ttcatggtat agcgctcatg gcattacact gaaagatggt ttgaaacgcc agttagccat
     2521 gcaccatgct tttgagtgta tgggcgaacc actcgcacgc gatttcgatg cgcagatgtt
     2581 ttcccgctac cgagaaaaac ggttaaaagg tgagtatgcc cgttcaaaca gagtgaaaga
     2641 ggtatcgcct cgcacgctta atcttgagct ggcctacttc cgggcggtgt tcaatgagct
     2701 aaaccgcctc ggagaatgga agggtgaaaa cccactaaaa aatatgcgcc cattccgcac
     2761 agaagaaatg gaaatgactt ggctaactca cgaccaaatt tcgcaactgc tcggagagtg
     2821 taaccgacat gaccaccctg atttagaaac cgtggtaaga atctgtctcg caactggcgc
     2881 acggtggtct gaggccgaga gtctgagaaa aagccaactc gcgaaataca aaatcacata
     2941 caccaacacg aaaggtagaa aaaatcgcac cgtcccaatc agcaaagagc tctatgagtc
     3001 tctgcctgat gataaaaagg gccggttgtt cagtgattgt tatggcgcgt tccggtcagc
     3061 tttggaaaga acaggtatcg aactaccggc aggacaactt acccacgttt tacgtcacac
     3121 cttcgctagt cactttatga tgaatggcgg taatattttg gtgttgcagc gcgtactcgg
     3181 ccatacagat ataaaaatga ccatgcgata tgcgcacttt gcacccgacc atttagagga
     3241 tgcagttaaa ctaaatcctt tagttcacat tactaacagc aaataagtta tggaggttgt
     3301 atgtcagttc ggctagccca tgggtatgga cagttttccc tttgatatgt aacgcacgtt
     3361 gtgcatcgat ttgacaccct atcattgata gagttatttt cccactccct atcagtgata
     3421 gagaaaagtg aaatgaatag ttcgacaaag atcgcattgg taattacgtt actcgatgcc
     3481 atggggattg gccttatcat gccagtcttg ccaacgttat tacgtgaatt tattgcttcg
     3541 gaagatatcg ctaaccactt tggcgtattg cttgcacttt atgcgttaat gcaggttatc
     3601 tttgctcctt ggcttggaaa aatgtctgac cgatttggtc ggcgcccagt gctgttgttg
     3661 tcattaatag gcgcatcgct ggattactta ttgctggctt tttcaagtgc gctttggatg
     3721 ctgtatttag gccgtttgct ttcagggatc acaggagcta ctggggctgt cgcggcatcg
     3781 gtcattgccg ataccacctc agcttctcaa cgcgtgaagt ggttcggttg gttaggggca
     3841 agttttgggc ttggtttaat agcggggcct attattggtg gttttgcagg agagatttca
     3901 ccgcatagtc ccttttttat cgctgcgttg ctaaatattg tcactttcct tgtggttatg
     3961 ttttggttcc gtgaaaccaa aaatacacgt gataatacag ataccgaagt aggggttgag
     4021 acgcaatcaa attcggtgta catcacttta tttaaaacga tgcccatttt gttgattatt
     4081 tatttttcag cgcaattgat aggccaaatt cccgcaacgg tgtgggtgct atttaccgaa
     4141 aatcgttttg gatggaatag catgatggtt ggcttttcat tagcgggtct tggtctttta
     4201 cactcagtat tccaagcctt tgtggcagga agaatagcca ctaaatgggg cgaaaaaacg
     4261 gcagtactgc tcgaatttat tgcagatagt agtgcatttg cctttttagc gtttatatct
     4321 gaaggttggt tagatttccc tgttttaatt ttattggctg gtggtgggat cgctttacct
     4381 gcattacagg gagtgatgtc tatccaaaca aagagtcatg agcaaggtgc tttacaggga
     4441 ttattggtga gccttaccaa tgcaaccggt gttattggcc cattactgtt tactgttatt
     4501 tataatcatt cactaccaat ttgggatggc tggatttgga ttattggttt agcgttttac
     4561 tgtattatta tcctgctatc aatgaccttc atgttgaccc ctcaagctca ggggagtaaa
     4621 caggagacaa gtgcttagtt atttcgtcac caaatgaggc ctccaaaact tcatttttaa
     4681 tttttgagct cgcttggact cctgttgata gatccagtaa tgacctcaga actccatctg
     4741 gatttgttca gaacgctcgg ttgccgccgg gcgtttttta ttggtgagaa tccaagcgtc
     4801 gacctagtgc ggccgcaaga tccggccacg atgcgtccgg cgtagaggat ctgaagatca
     4861 gcagttcaac ctgttgatag tacgtactaa gctctcatgt ttcacgtact aagctctcat
     4921 gtttaacgta ctaagctctc atgtttaacg aactaaaccc tcatggctaa cgtactaagc
     4981 tctcatggct aacgtactaa gctctcatgt ttcacgtact aagctctcat gtttgaacaa
     5041 taaaattaat ataaatcagc aacttaaata gcctctaagg ttttaagttt tataagaaaa
     5101 aaaagaatat ataaggcttt taaagctttt aaggtttaac ggttgtggac aacaagccag
     5161 ggatgtaacg cactgagaag cccttagagc ctctcaaagc aattttgagt gacacaggaa
     5221 cacttaacgg ctgacatggg aattaggaag ttcctattcc gaagttccta ttctctagaa
     5281 agtataggaa cttccatatg ccatgggcga tggttctgag taacagataa tagaatggcg
     5341 ataaagtggc ggtagaaatg gcgaataata ggtaattatt ggcaaacaat ggcaatctat
     5401 gtcaatgata aataacgcat actattgatt ttcggttgtt cttgtaggaa ctcataatcg
     5461 cttggtcgct ggttcaagtc cagcaggggc caccaaattt aagtagtaaa tacatacagt
     5521 taggccactc tctcgagtgg cctttttgtt gcctcgtttt cgagtggcga tagaatggcg
     5581 gtggattttt taccgccact ttcctgagtg catgctagct tcttcgtctg tttctactgg
     5641 tattggcaca aacctgattc caatttgagc aaggctatgt gccatctcga tactcgttct
     5701 taactcaaca gaagatgctt tgtgcataca gcccctcgtt tattatttat ctcctcagcc
     5761 agccgctgtg ctttcagtgg atttcggata acagaaaggc cgggaaatac ccagcctcgc
     5821 tttgtaacgg agtagagacg aaagtgattg cgcctacccg gatattatcg tgaggatgcg
     5881 tcatcgccat taattcactg atcagtgata gctgtcaaac atgagaattg atccggctgc
     5941 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     6001 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     6061 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatgct
     6121 gcacatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcaagaatt
     6181 aattcccaat tccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     6241 ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga
     6301 tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc cataaactgc
     6361 caggaattaa ttccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     6421 gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg
     6481 atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg
     6541 ccaggaatta attccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
     6601 cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg
     6661 gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc gccataaact
     6721 gccaggaatt aattccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     6781 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc
     6841 ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac
     6901 tgccaggaat tggggatcg
//
