LOCUS       Exported                6919 bp ds-DNA     circular SYN 18-DEC-2018
DEFINITION  Chromosomal integration vector pOSIP-TO, complete sequence.
ACCESSION   KF030466
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)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6919
                     /organism="Chromosomal integration vector pOSIP-TO"
                     /mol_type="other DNA"
                     /db_xref="taxon:1340187"
     misc_feature    1..1170
                     /note="multiple cloning site (MCS) containing ccdB positive
                     selection marker and pUC origin; parental vector must be 
                     propagated in permissive strain DB3.1"
     CDS             142..447
                     /codon_start=1
                     /product="controlled cell death protein B"
                     /label=controlled cell death protein B
                     /note="CcdB"
                     /protein_id="AGN12528.1"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     rep_origin      451..1124
                     /note="high copy replication origin derived from pUC19 
                     accession M77789 (BBa_I50022)"
     rep_origin      477..1065
                     /direction=RIGHT
                     /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"
     regulatory      1170..1231
                     /regulatory_class="terminator"
                     /note="rrnBT2 terminator from E.coli, forward orientation"
     terminator      1200..1227
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     regulatory      1247..1338
                     /regulatory_class="terminator"
                     /note="tonB terminator from E.coli, reverse orientation"
     terminator      1276..1307
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     misc_feature    1384..1417
                     /note="FRT; Flp recombinase target site"
     protein_bind    1384..1417
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     CDS             complement(1424..2137)
                     /codon_start=1
                     /product="lambda temperature sensitive repressor"
                     /label=lambda temperature sensitive repressor
                     /note="CIts"
                     /protein_id="AGN12529.1"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPKLRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     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
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2276..3286
                     /codon_start=1
                     /product="phage 186 integrase protein"
                     /label=phage 186 integrase protein
                     /protein_id="AGN12530.1"
                     /translation="MTVRKNPAGGWICELYPNGAKGKRIRKKFATKGEALAFEQYTVQN
                     PWQEEKEDRRTLKELVDSWYSAHGITLKDGLKRQLAMHHAFECMGEPLARDFDAQMFSR
                     YREKRLKGEYARSNRVKEVSPRTLNLELAYFRAVFNELNRLGEWKGENPLKNMRPFRTE
                     EMEMTWLTHDQISQLLGECNRHDHPDLETVVRICLATGARWSEAESLRKSQLAKYKITY
                     TNTKGRKNRTVPISKELYESLPDDKKGRLFSDCYGAFRSALERTGIELPAGQLTHVLRH
                     TFASHFMMNGGNILVLQRVLGHTDIKMTMRYAHFAPDHLEDAVKLNPLVHITNSK"
     promoter        3371..3425
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    3406..3424
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             3433..4638
                     /codon_start=1
                     /product="tetracyclin resistance protein"
                     /label=tetracyclin resistance protein
                     /protein_id="AGN12531.1"
                     /translation="MNSSTKIALVITLLDAMGIGLIMPVLPTLLREFIASEDIANHFGV
                     LLALYALMQVIFAPWLGKMSDRFGRRPVLLLSLIGASLDYLLLAFSSALWMLYLGRLLS
                     GITGATGAVAASVIADTTSASQRVKWFGWLGASFGLGLIAGPIIGGFAGEISPHSPFFI
                     AALLNIVTFLVVMFWFRETKNTRDNTDTEVGVETQSNSVYITLFKTMPILLIIYFSAQL
                     IGQIPATVWVLFTENRFGWNSMMVGFSLAGLGLLHSVFQAFVAGRIATKWGEKTAVLLE
                     FIADSSAFAFLAFISEGWLDFPVLILLAGGGIALPALQGVMSIQTKSHEQGALQGLLVS
                     LTNATGVIGPLLFTVIYNHSLPIWDGWIWIIGLAFYCIIILLSMTFMLTPQAQGSKQET
                     SA"
     terminator      4697..4791
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      4817..5246
                     /note="R6Kgamma; pir dependent origin of replication"
     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)."
     misc_recomb     5307..5613
                     /label=phage 186 attP site
                     /note="phage 186 attP site"
     protein_bind    5307..5521
                     /gene="attP"
                     /label=phage 186 attP
                     /bound_moiety="phage 186 integrase"
                     /note="attachment site of phage 186"
     regulatory      5620..5891
                     /regulatory_class="terminator"
                     /note="lambda tL3 terminator"
     terminator      5634..5880
                     /label=-lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     regulatory      6194..6905
                     /regulatory_class="terminator"
                     /note="rrnBT1 terminator from E.coli; 4 copies"
     terminator      6200..6286
                     /gene="Escherichia coli rrnB"
                     /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      6743..6829
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"