LOCUS       40924_33927        6925 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Chromosomal integration vector pOSIP-TH, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6925)
  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 6925)
  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 6925)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6925)
  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..6925
                     /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             2270..3340
                     /label=HK022 integrase
                     /note="integrase from phage HK022"
     protein_bind    3461..3479
                     /label=tet operator
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             3488..4690
                     /label=TcR
                     /note="tetracycline efflux protein"
     terminator      4752..4846
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      4903..5291
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     misc_feature    5302..5349
                     /note="FRT; Flp recombinase target site"
     protein_bind    5302..5349
                     /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(5379..5605)
                     /label=HK022 attP
                     /note="attachment site of phage HK022"
     terminator      complement(5640..5886)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     terminator      6206..6292
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      6387..6473
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      6568..6654
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      complement(6752..6795)
                     /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 cgcgtgtagc cttttgaaga ggatcagaaa tgggaagaag
     2281 gcgaagtcat gagcgccggg atttaccccc taacctttat ataagaaaca atggatatta
     2341 ctgctacagg gacccaagga cgggtaaaga gtttggatta ggccgagaca ggcgaatcgc
     2401 aatcactgaa gctatacagg ccaacattga gttgctatcc gggaacaggc gtgagtcact
     2461 gatagacaga attaaaggcg ctgacgcaat cactcttcat gcgtggcttg accgatatga
     2521 aacaatcctc agcgagaggg gtatcaggcc gaaaactcta ctcgactacg ccagcaaaat
     2581 cagagcaatc cgaagaaaat tgccggacaa accgctcgct gacatatcaa cgaaagaggt
     2641 ggcagcaatg ctaaacacct acgtagcaga aggcaaatcg gcttccgcaa aattgatcag
     2701 gtcaaccctt gttgacgttt ttcgtgaggc aatagccgag gggcatgtgg ctacgaatcc
     2761 ggtaacagca acccgcacag caaaatcaga agtaaggcgt tcaagactga cagctaatga
     2821 atatgtcgcg atataccatg cagccgaacc gctcccaatc tggctgagac tggcaatgga
     2881 tttggctgtc gttacagggc agagagtagg cgatttgtgc agaatgaaat ggtcagacat
     2941 aaacgacaac catcttcaca ttgaacagag taaaacaggg gctaaactcg ccattccact
     3001 aacgctaacg attgacgcgc tcaatatctc attggctgat acactacagc aatgcaggga
     3061 ggccagcagc agtgaaacta taatcgcatc aaagcatcac gatccgcttt ccccgaaaac
     3121 agtatcaaag tattttacaa aggcgagaaa tgcatctgga ctctcatttg atggaaaccc
     3181 gccaacattc catgaactgc gtagcctgtc agcgaggcta taccggaacc agattggcga
     3241 taagtttgct caacgtcttc tcgggcataa atcagattca atggcggcgc ggtataggga
     3301 cagccgtgga cgggaatggg acaaaattga aatcgacaaa tgattttatt ttgactaata
     3361 atgacctact taccatgggt atggacagtt ttccctttga tatgtaacgc acgttgtgca
     3421 tcgatttgac accctatcat tgatagagtt attttcccac tccctatcag tgatagagaa
     3481 aagtgaaatg aatagttcga caaagatcgc attggtaatt acgttactcg atgccatggg
     3541 gattggcctt atcatgccag tcttgccaac gttattacgt gaatttattg cttcggaaga
     3601 tatcgctaac cactttggcg tattgcttgc actttatgcg ttaatgcagg ttatctttgc
     3661 tccttggctt ggaaaaatgt ctgaccgatt tggtcggcgc ccagtgctgt tgttgtcatt
     3721 aataggcgca tcgctggatt acttattgct ggctttttca agtgcgcttt ggatgctgta
     3781 tttaggccgt ttgctttcag ggatcacagg agctactggg gctgtcgcgg catcggtcat
     3841 tgccgatacc acctcagctt ctcaacgcgt gaagtggttc ggttggttag gggcaagttt
     3901 tgggcttggt ttaatagcgg ggcctattat tggtggtttt gcaggagaga tttcaccgca
     3961 tagtcccttt tttatcgctg cgttgctaaa tattgtcact ttccttgtgg ttatgttttg
     4021 gttccgtgaa accaaaaata cacgtgataa tacagatacc gaagtagggg ttgagacgca
     4081 atcaaattcg gtgtacatca ctttatttaa aacgatgccc attttgttga ttatttattt
     4141 ttcagcgcaa ttgataggcc aaattcccgc aacggtgtgg gtgctattta ccgaaaatcg
     4201 ttttggatgg aatagcatga tggttggctt ttcattagcg ggtcttggtc ttttacactc
     4261 agtattccaa gcctttgtgg caggaagaat agccactaaa tggggcgaaa aaacggcagt
     4321 actgctcgaa tttattgcag atagtagtgc atttgccttt ttagcgttta tatctgaagg
     4381 ttggttagat ttccctgttt taattttatt ggctggtggt gggatcgctt tacctgcatt
     4441 acagggagtg atgtctatcc aaacaaagag tcatgagcaa ggtgctttac agggattatt
     4501 ggtgagcctt accaatgcaa ccggtgttat tggcccatta ctgtttactg ttatttataa
     4561 tcattcacta ccaatttggg atggctggat ttggattatt ggtttagcgt tttactgtat
     4621 tattatcctg ctatcaatga ccttcatgtt gacccctcaa gctcagggga gtaaacagga
     4681 gacaagtgct tagttatttc gtcaccaaat gaggcctcca aaacttcatt tttaattttt
     4741 gagctcgctt ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt
     4801 gttcagaacg ctcggttgcc gccgggcgtt ttttattggt gagaatccaa gcgtcgacct
     4861 agtgcggccg caagatccgg ccacgatgcg tccggcgtag aggatctgaa gatcagcagt
     4921 tcaacctgtt gatagtacgt actaagctct catgtttcac gtactaagct ctcatgttta
     4981 acgtactaag ctctcatgtt taacgaacta aaccctcatg gctaacgtac taagctctca
     5041 tggctaacgt actaagctct catgtttcac gtactaagct ctcatgtttg aacaataaaa
     5101 ttaatataaa tcagcaactt aaatagcctc taaggtttta agttttataa gaaaaaaaag
     5161 aatatataag gcttttaaag cttttaaggt ttaacggttg tggacaacaa gccagggatg
     5221 taacgcactg agaagccctt agagcctctc aaagcaattt tgagtgacac aggaacactt
     5281 aacggctgac atgggaatta ggaagttcct attccgaagt tcctattctc tagaaagtat
     5341 aggaacttcc atatgccatg ggacaaaatt gaaatcgaca aatgatttta ttttgactaa
     5401 taatgaccta cttacattaa tttactgata attaaagaga ttttaaatat acaacttatt
     5461 cacctaaagg atgacaaaat aacattaatc acttaaaaat catcgcatta cactaatctg
     5521 tggttaaatg atagactaca taatgcgaca aaacgcaaca tatccagtca ctatgaatca
     5581 actacttaga tagtattagt gacctgagac agagcattag ctagcttctt cgtctgtttc
     5641 tactggtatt ggcacaaacc tgattccaat ttgagcaagg ctatgtgcca tctcgatact
     5701 cgttcttaac tcaacagaag atgctttgtg catacagccc ctcgtttatt atttatctcc
     5761 tcagccagcc gctgtgcttt cagtggattt cggataacag aaaggccggg aaatacccag
     5821 cctcgctttg taacggagta gagacgaaag tgattgcgcc tacccggata ttatcgtgag
     5881 gatgcgtcat cgccattaat tcactgatca gtgatagctg tcaaacatga gaattgatcc
     5941 ggctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     6001 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     6061 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     6121 tatgctgcac atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca
     6181 agaattaatt cccaattcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     6241 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg
     6301 agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata
     6361 aactgccagg aattaattcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     6421 cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg
     6481 gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat
     6541 aaactgccag gaattaattc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg
     6601 gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg
     6661 ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca
     6721 taaactgcca ggaattaatt ccaggcatca aataaaacga aaggctcagt cgaaagactg
     6781 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
     6841 gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc
     6901 ataaactgcc aggaattggg gatcg
//
