LOCUS       Exported                6298 bp ds-DNA     circular SYN 29-AUG-2018
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pOSIP-KO (KanR, 186)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6298)
  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 Sep 20;2(9):537-41. doi: 10.1021/sb400021j. 
            Epub 2013 May 20.
  PUBMED    24050148
REFERENCE   2  (bases 1 to 6298)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 29, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6298
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             142..447
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     326..345
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     rep_origin      477..1065
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     966..985
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     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      1276..1307
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     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
                     /gene="cIts"
                     /product="temperature-sensitive variant of the phage lambda
                     repressor"
                     /label=-lambda repressor (ts)
                     /note="thermosensitivity is conferred by the A67T mutation"
                     /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
                     /gene="int"
                     /product="integrase from phage 186"
                     /label=phage 186 integrase
                     /translation="MTVRKNPAGGWICELYPNGAKGKRIRKKFATKGEALAFEQYTVQN
                     PWQEEKEDRRTLKGLVDSWYSAHGITLKDGLKRQLAMHHAFECMGEPLARDFDAQMFSR
                     YREKRLKGEYARSNRVKEVSPRTLNLELAYFRAVFNELNRLGEWKGENPLKNMRPFRTE
                     EMEMTWLTHDQISQLLGECNRHDHPDLETVVRICLATGARWSEAESLRKSQLAKYKITY
                     TNTKGRKNRTVPISKELYESLPDDKKGRLFSDCYGAFRSALERTGIELPAGQLTHVLRH
                     TFASHFMMNGGNILVLQRVLGHTDIKMTMRYAHFAPDHLEDAVKLNPLVHITNSK"
     primer_bind     complement(3449..3468)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3460..4275
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3533..3552)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4397..4785
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    4796..4843
                     /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    4856..5070
                     /gene="attP"
                     /label=phage 186 attP
                     /bound_moiety="phage 186 integrase"
                     /note="attachment site of phage 186"
     terminator      5187..5433
                     /label=-lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     primer_bind     complement(5524..5546)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5697..5715
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      5756..5842
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5939..6025
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6122..6208
                     /gene="Escherichia coli rrnB"
                     /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 ggctggttga
     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 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
     3421 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     3481 acgtcttgct cccgtccgcg cttaaactcc aacatggacg ctgatttata tgggtataaa
     3541 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta tgggaagccc
     3601 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     3661 gagatggtcc gtctcaactg gctgacggag tttatgcctc tcccgaccat caagcatttt
     3721 atccgtactc ctgatgatgc gtggttactc accaccgcga ttcctgggaa aacagccttc
     3781 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggccgtgttc
     3841 ctgcgccggt tacattcgat tcctgtttgt aattgtcctt ttaacagcga tcgtgtattt
     3901 cgtcttgctc aggcgcaatc acgcatgaat aacggtttgg ttgatgcgag tgattttgat
     3961 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcacaa gctcttgcca
     4021 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     4081 gaggggaaat taataggttg tattgatgtt ggacgggtcg gaatcgcaga ccgttaccag
     4141 gaccttgcca ttctttggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     4201 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     4261 gatgagtttt tctaataata ctagcagaaa tcatccttag cgaaagctaa ggattttttt
     4321 tatctgatta ccgcctttga gtgagcgtcg acctagtgcg gccgcaagat ccggccacga
     4381 tgcgtccggc gtagaggatc tgaagatcag cagttcaacc tgttgatagt acgtactaag
     4441 ctctcatgtt tcacgtacta agctctcatg tttaacgtac taagctctca tgtttaacga
     4501 actaaaccct catggctaac gtactaagct ctcatggcta acgtactaag ctctcatgtt
     4561 tcacgtacta agctctcatg tttgaacaat aaaattaata taaatcagca acttaaatag
     4621 cctctaaggt tttaagtttt ataagaaaaa aaagaatata taaggctttt aaagctttta
     4681 aggtttaacg gttgtggaca acaagccagg gatgtaacgc actgagaagc ccttagagcc
     4741 tctcaaagca attttgagtg acacaggaac acttaacggc tgacatggga attaggaagt
     4801 tcctattccg aagttcctat tctctagaaa gtataggaac ttccatatgc catgggcgat
     4861 ggttctgagt aacagataat agaatggcga taaagtggcg gtagaaatgg cgaataatag
     4921 gtaattattg gcaaacaatg gcaatctatg tcaatgataa ataacgcata ctattgattt
     4981 tcggttgttc ttgtaggaac tcataatcgc ttggtcgctg gttcaagtcc agcaggggcc
     5041 accaaattta agtagtaaat acatacagtt aggccactct ctcgagtggc ctttttgttg
     5101 cctcgttttc gagtggcgat agaatggcgg tggwtttttt accgccactt tcctgaggag
     5161 tgcatagcta gcttcttcgt ctgtttctac tggtattggc acaaacctga ttccaatttg
     5221 agcaaggcta tgtgccatct cgatactcgt tcttaactca acagaagatg ctttgtgcat
     5281 acagcccctc gtttattatt tatctcctca gccagccgct gtgctttcag tggatttcgg
     5341 ataacagaaa ggccgggaaa tacccagcct cgctttgtaa cggagtagag acgaaagtga
     5401 ttgcgcctac ccggatatta tcgtgaggat gcgtcatcgc cattaattca ctgatcagtg
     5461 ataagctgtc aaacatgaga attgatccgg ctgcctcgcg cgtttcggtg atgacggtga
     5521 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     5581 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     5641 gacccagtca cgtagcgata gcggagtgta tgctgcacat gacattaacc tataaaaata
     5701 ggcgtatcac gaggcccttt cgtcttcaag aattaattcc caattcccca ggcatcaaat
     5761 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     5821 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc
     5881 cggagggtgg cgggcaggac gcccgccata aactgccagg aattaattcc ccaggcatca
     5941 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     6001 gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg
     6061 gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggaattaat tccccaggca
     6121 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
     6181 ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca
     6241 acggcccgga gggtggcggg caggacgccc gccataaact gccaggaatt ggggatcg
//
