LOCUS       pOSIP-KC.        6970 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Prokaryotic one-step cloning and chromosomal integration vector 
            encoding a kanamycin resistance marker and the phage phi-C31 
            integrase.
ACCESSION   .
VERSION     .
KEYWORDS    pOSIP-KC
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6970)
  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 2013;2:537-41.
  PUBMED    24050148
REFERENCE   2  (bases 1 to 6970)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6970)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol"; date: "2013"; volume: "2"; pages: "537-41"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     Because of the toxic ccdB gene, this vector must be propagated in a 
            permissive strain such as DB3.1(TM).
FEATURES             Location/Qualifiers
     source          1..6970
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..75
                     /label=MCS 1
                     /note="MCS 1"
                     /note="multiple cloning site, part 1"
     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"
     CDS             2272..4086
                     /label=phage phi-C31 integrase
                     /note="integrase from phage phi-C31"
     CDS             4224..5036
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     rep_origin      5161..5549
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     protein_bind    5560..5607
                     /label=FLP recombinase from the Saccharomyces
                     cerevisi
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="FRT"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     protein_bind    complement(5676..5775)
                     /label=phage phi-C31 attP
                     /note="attachment site of phage phi-C31"
     terminator      complement(5860..6106)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     terminator      6428..6514
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      6611..6697
                     /gene="Escherichia coli rrnB"
                     /label=Escherichia coli rrnB terminator
                     /note="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      complement(6797..6840)
                     /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 aggttgtatc ctgcaggccg agaaaaagga ggagataaac agtggacacg
     2281 tacgcgggtg cttacgaccg tcagtcgcgc gagcgcgaga attcgagcgc agcaagccca
     2341 gcgacacagc gtagcgccaa cgaagacaag gcggccgacc ttcagcgcga agtcgagcgc
     2401 gacgggggcc ggttcaggtt cgtcgggcat ttcagcgaag cgccgggcac gtcggcgttc
     2461 gggacggcgg agcgcccgga gttcgaacgc atcctgaacg aatgccgcgc cgggcggctc
     2521 aacatgatca ttgtctatga cgtgtcgcgc ttctcgcgcc tgaaggtcat ggacgcgatt
     2581 ccgattgtct cggaattgct cgccctgggc gtgacgattg tttccactca ggaaggcgtc
     2641 ttccggcagg gaaacgtcat ggacctgatt cacctgatta tgcggctcga cgcgtcgcac
     2701 aaagaatctt cgctgaagtc ggcgaagatt ctcgacacga agaaccttca gcgcgaattg
     2761 ggcgggtacg tcggcgggaa ggcgccttac ggcttcgagc ttgtttcgga gacgaaggag
     2821 atcacgcgca acggccgaat ggtcaatgtc gtcatcaaca agcttgcgca ctcgaccact
     2881 ccccttaccg gacccttcga gttcgagccc gacgtaatcc ggtggtggtg gcgtgagatc
     2941 aagacgcaca aacaccttcc cttcaagccg ggcagtcaag ccgccattca cccgggcagc
     3001 atcacggggc tttgtaagcg catggacgct gacgccgtgc cgacccgggg cgagacgatt
     3061 gggaagaaga ccgcttcaag cgcctgggac ccggcaaccg ttatgcgaat ccttcgggac
     3121 ccgcgtattg cgggcttcgc cgctgaggtg atctacaaga agaagccgga cggcacgccg
     3181 accacgaaga ttgagggtta ccgcattcag cgcgacccga tcacgctccg gccggtcgag
     3241 cttgattgcg gaccgatcat cgagcccgct gagtggtatg agcttcaggc gtggttggac
     3301 ggcagggggc gcggcaaggg gctttcccgg gggcaagcca ttctgtccgc catggacaag
     3361 ctgtactgcg agtgtggcgc cgtcatgact tcgaagcgcg gggaagaatc gatcaaggac
     3421 tcttaccgct gccgtcgccg gaaggtggtc gacccgtccg cacctgggca gcacgaaggc
     3481 acgtgcaacg tcagcatggc ggcactcgac aagttcgttg cggaacgcat cttcaacaag
     3541 atcaggcacg ccgaaggcga cgaagagacg ttggcgcttc tgtgggaagc cgcccgacgc
     3601 ttcggcaagc tcactgaggc gcctgagaag agcggcgaac gggcgaacct tgttgcggag
     3661 cgcgccgacg ccctgaacgc ccttgaagag ctgtacgaag accgcgcggc aggcgcgtac
     3721 gacggacccg ttggcaggaa gcacttccgg aagcaacagg cagcgctgac gctccggcag
     3781 caaggggcgg aagagcggct tgccgaactt gaagccgccg aagccccgaa gcttcccctt
     3841 gaccaatggt tccccgaaga cgccgacgct gacccgaccg gccctaagtc gtggtggggg
     3901 cgcgcgtcag tagacgacaa gcgcgtgttc gtcgggctct tcgtagacaa gatcgttgtc
     3961 acgaagtcga ctacgggcag ggggcaggga acgcccatcg agaagcgcgc ttcgatcacg
     4021 tgggcgaagc cgccgaccga cgacgacgaa gacgacgccc aggacggcac ggaagacgta
     4081 gcggcgtagt tttattttga ctaataatga cctacttaca cgttgtgtct caaaatctct
     4141 gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca
     4201 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctcccgtc
     4261 cgcgcttaaa ctccaacatg gacgctgatt tatatgggta taaatgggct cgcgataatg
     4321 tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt
     4381 ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtccgtctca
     4441 actggctgac ggagtttatg cctctcccga ccatcaagca ttttatccgt actcctgatg
     4501 atgcgtggtt actcaccacc gcgattcctg ggaaaacagc cttccaggta ttagaagaat
     4561 atcctgattc aggtgaaaat attgttgatg cgctggccgt gttcctgcgc cggttacatt
     4621 cgattcctgt ttgtaattgt ccttttaaca gcgatcgtgt atttcgtctt gctcaggcgc
     4681 aatcacgcat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct
     4741 ggcctgttga acaagtctgg aaagaaatgc acaagctctt gccattctca ccggattcag
     4801 tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag
     4861 gttgtattga tgttggacgg gtcggaatcg cagaccgtta ccaggacctt gccattcttt
     4921 ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta
     4981 ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat
     5041 aatactagca gaaatcatcc ttagcgaaag ctaaggattt tttttatctg attaccgcct
     5101 ttgagtgagc gtcgacctag tgcggccgca agatccggcc acgatgcgtc cggcgtagag
     5161 gatctgaaga tcagcagttc aacctgttga tagtacgtac taagctctca tgtttcacgt
     5221 actaagctct catgtttaac gtactaagct ctcatgttta acgaactaaa ccctcatggc
     5281 taacgtacta agctctcatg gctaacgtac taagctctca tgtttcacgt actaagctct
     5341 catgtttgaa caataaaatt aatataaatc agcaacttaa atagcctcta aggttttaag
     5401 ttttataaga aaaaaaagaa tatataaggc ttttaaagct tttaaggttt aacggttgtg
     5461 gacaacaagc cagggatgta acgcactgag aagcccttag agcctctcaa agcaattttg
     5521 agtgacacag gaacacttaa cggctgacat gggaattagg aagttcctat tccgaagttc
     5581 ctattctcta gaaagtatag gaacttccat atgccatggt cgcgctcgcg cgactgacgg
     5641 tcgtaagcac ccgcgtacgt gtccaccccg gtcacaaccc cttgtgtcat gtcggcgacc
     5701 ctacgccccc aactgagaga actcaaaggt taccccagtt ggggcactac tcccgaaaac
     5761 cgcttctgac ctgggaaaac gtgaagcccc ggggcatccg ctgagggttg ccgccggggc
     5821 ttcggtgtgt ccgtcagtag ctagcttctt cgtctgtttc tactggtatt ggcacaaacc
     5881 tgattccaat ttgagcaagg ctatgtgcca tctcgatact cgttcttaac tcaacagaag
     5941 atgctttgtg catacagccc ctcgtttatt atttatctcc tcagccagcc gctgtgcttt
     6001 cagtggattt cggataacag aaaggccggg aaatacccag cctcgctttg taacggagta
     6061 gagacgaaag tgattgcgcc tacccggata ttatcgtgag gatgcgtcat cgccattaat
     6121 tcactgatca gtgatagctg tcaaacatga gaattgatcc ggctgcctcg cgcgtttcgg
     6181 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     6241 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     6301 gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatgctgcac atgacattaa
     6361 cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattaatt cccaattccc
     6421 caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
     6481 tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc
     6541 gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca ggaattaatt
     6601 ccccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     6661 ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt
     6721 tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg ccataaactg ccaggaatta
     6781 attccccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat
     6841 ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa
     6901 cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggaa
     6961 ttggggatcg
//
