LOCUS       40924_33907        6764 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Chromosomal integration vector pOSIP-KL, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6764)
  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 6764)
  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 6764)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6764)
  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..6764
                     /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)"
     CDS             2379..3446
                     /label=lambda integrase
                     /note="integrase from phage lambda"
     CDS             3676..4488
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      4613..5001
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     protein_bind    5012..5059
                     /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)."
     misc_feature    5026..5059
                     /note="FRT; Flp recombinase target site"
     protein_bind    5122..5355
                     /label=lambda attP
                     /note="integrase from phage lambda"
     terminator      complement(5479..5725)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     terminator      6045..6131
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      6226..6312
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      6407..6493
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      complement(6591..6634)
                     /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 cgcgtcgtcg atgggttcgg gaatgcagga tattcccacc
     2281 tccggttaag gatggaagag agtatctgtt ccacgaatca gcggtaaagg ttgacttaaa
     2341 tcgaccagta acaggtggcc ttttgaagag gatcagaaat gggaagaagg cgaagtcatg
     2401 agcgccggga tttaccccct aacctttata taagaaacaa tggatattac tgctacaggg
     2461 acccaaggac gggtaaagag tttggattag gcagagacag gcgaatcgca atcactgaag
     2521 ctatacaggc caacattgag ttattttcag gacacaaaca caagcctctg acagcgagaa
     2581 tcaacagtga taattccgtt acgttacatt catggcttga tcgctacgaa aaaatcctgg
     2641 ccagcagagg aatcaagcag aagacactca taaattacat gagcaaaatt aaagcaataa
     2701 ggaggggtct gcctgatgct ccacttgaag acatcaccac aaaagaaatt gcggcaatgc
     2761 tcaatggata catagacgag ggcaaggcgg cgtcagccaa gttaatcaga tcaacactga
     2821 gcgatgcatt ccgagaggca atagctgaag gccatataac aacaaaccat gtcgctgcca
     2881 ctcgcgcagc aaaatcagag gtaaggagat caagacttac ggctgacgaa tacctgaaaa
     2941 tttatcaagc agcagaatca tcaccatgtt ggctcagact tgcaatggaa ctggctgttg
     3001 ttaccgggca acgagttggt gatttatgcg aaatgaagtg gtctgatatc gtagatggat
     3061 atctttatgt cgagcaaagc aaaacaggcg taaaaattgc catcccaaca gcattgcata
     3121 ttgatgctct cggaatatca atgaaggaaa cacttgataa atgcaaagag attcttggcg
     3181 gagaaaccat aattgcatct actcgtcgcg aaccgctttc atccggcaca gtatcaaggt
     3241 attttatgcg cgcacgaaaa gcatcaggtc tttccttcga aggggatccg cctacctttc
     3301 acgagttgcg cagtttgtct gcaagactct atgagaagca gataagcgat aagtttgctc
     3361 aacatcttct cgggcataag tcggacacca tggcatcaca gtatcgtgat gacagaggca
     3421 gggagtggga caaaattgaa atcaaataat gattttattt tgactgatag tgacctgttc
     3481 gttgcaacaa attgataagc aatgccaagc ttggcactgg ctgatcagct agcccatggg
     3541 tatggacagt tttccctttg atatgtaacg cacgttgtgt ctcaaaatct ctgatgttac
     3601 attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta cataaacagt
     3661 aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctcccg tccgcgctta
     3721 aactccaaca tggacgctga tttatatggg tataaatggg ctcgcgataa tgtcgggcaa
     3781 tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt gtttctgaaa
     3841 catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtccgtct caactggctg
     3901 acggagttta tgcctctccc gaccatcaag cattttatcc gtactcctga tgatgcgtgg
     3961 ttactcacca ccgcgattcc tgggaaaaca gccttccagg tattagaaga atatcctgat
     4021 tcaggtgaaa atattgttga tgcgctggcc gtgttcctgc gccggttaca ttcgattcct
     4081 gtttgtaatt gtccttttaa cagcgatcgt gtatttcgtc ttgctcaggc gcaatcacgc
     4141 atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt
     4201 gaacaagtct ggaaagaaat gcacaagctc ttgccattct caccggattc agtcgtcact
     4261 catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat aggttgtatt
     4321 gatgttggac gggtcggaat cgcagaccgt taccaggacc ttgccattct ttggaactgc
     4381 ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg tattgataat
     4441 cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta ataatactag
     4501 cagaaatcat ccttagcgaa agctaaggat tttttttatc tgattaccgc ctttgagtga
     4561 gcgtcgacct agtgcggccg caagatccgg ccacgatgcg tccggcgtag aggatctgaa
     4621 gatcagcagt tcaacctgtt gatagtacgt actaagctct catgtttcac gtactaagct
     4681 ctcatgttta acgtactaag ctctcatgtt taacgaacta aaccctcatg gctaacgtac
     4741 taagctctca tggctaacgt actaagctct catgtttcac gtactaagct ctcatgtttg
     4801 aacaataaaa ttaatataaa tcagcaactt aaatagcctc taaggtttta agttttataa
     4861 gaaaaaaaag aatatataag gcttttaaag cttttaaggt ttaacggttg tggacaacaa
     4921 gccagggatg taacgcactg agaagccctt agagcctctc aaagcaattt tgagtgacac
     4981 aggaacactt aacggctgac atgggaatta ggaagttcct attccgaagt tcctattctc
     5041 tagaaagtat aggaacttcc atatgccatg gcatcacagt atcgtgatga cagaggcagg
     5101 gagtgggaca aaattgaaat caaataatga ttttattttg actgatagtg acctgttcgt
     5161 tgcaacaaat tgataagcaa tgctttttta taatgccaac ttagtataaa aaagctgaac
     5221 gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga
     5281 ctacataata ctgtaaaaca caacatatat gcagtcacta tgaatcaact acttagatgg
     5341 tattagtgac ctgtaacaga gcattagcgc aaggtgattt ttgtcttctt gcgctaattt
     5401 tttgtcatca aacctgtcgc actccagaga agcacaaagc ctcgcaatcc agtgcaaagc
     5461 tagcttcttc gtctgtttct actggtattg gcacaaacct gattccaatt tgagcaaggc
     5521 tatgtgccat ctcgatactc gttcttaact caacagaaga tgctttgtgc atacagcccc
     5581 tcgtttatta tttatctcct cagccagccg ctgtgctttc agtggatttc ggataacaga
     5641 aaggccggga aatacccagc ctcgctttgt aacggagtag agacgaaagt gattgcgcct
     5701 acccggatat tatcgtgagg atgcgtcatc gccattaatt cactgatcag tgatagctgt
     5761 caaacatgag aattgatccg gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
     5821 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     5881 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
     5941 acgtagcgat agcggagtgt atgctgcaca tgacattaac ctataaaaat aggcgtatca
     6001 cgaggccctt tcgtcttcaa gaattaattc ccaattccag gcatcaaata aaacgaaagg
     6061 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga
     6121 gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc
     6181 gggcaggacg cccgccataa actgccagga attaattcca ggcatcaaat aaaacgaaag
     6241 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     6301 agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg
     6361 cgggcaggac gcccgccata aactgccagg aattaattcc aggcatcaaa taaaacgaaa
     6421 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     6481 gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg
     6541 gcgggcagga cgcccgccat aaactgccag gaattaattc caggcatcaa ataaaacgaa
     6601 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc
     6661 tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt
     6721 ggcgggcagg acgcccgcca taaactgcca ggaattgggg atcg
//
