LOCUS       40924_36643        8696 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-23 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8696)
  AUTHORS   Saisho T, Shibahara K, Nakao A, Tanaka Y, Nishimura K, Kimura T, 
            Nakagawa T.
  TITLE     Improved Gateway Recycling Cloning System for Multigene Construction
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8696)
  AUTHORS   Saisho T, Shibahara K, Nakao A, Tanaka Y, Nishimura K, Kimura T, 
            Nakagawa T.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-NOV-2015) Contact:Tsuyoshi Nakagawa Shimane 
            University, Interdisciplinary Center for Science Research; 1060 
            Nishikawatsu, Matsue, Shimane 690-8504, Japan
REFERENCE   3  (bases 1 to 8696)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8696)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (02-NOV-2015) Contact:Tsuyoshi Nakagawa Shimane University, 
            Interdisciplinary Center for Science Research; 1060 Nishikawatsu, 
            Matsue, Shimane 690-8504, Japan"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     constructed using pDONR201.
FEATURES             Location/Qualifiers
     source          1..8696
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    28..127
                     /label=attL1
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    674..1071
                     /label=MD8
                     /note="MD8"
     promoter        1192..1375
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     protein_bind    1394..1518
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1555..1585
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1639..2295
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             2640..2942
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(2986..3110)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3121..3774
                     /codon_start=1
                     /label=GST
                     /note="glutathione S-transferase from Schistosoma
                     japonicum"
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             3781..3798
                     /codon_start=1
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
     terminator      3820..4072
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4281..4405
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4430..4460
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4732..4772
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4864..5721)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5722..5826)
                     /label=AmpR promoter
     protein_bind    complement(6123..6246)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6572..6667)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6791..7597
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7743..8331
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(8466..8493)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8585..8671)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
ORIGIN
        1 gttaacgcta gcatggatct cgggccccaa ataatgattt tattttgact gatagtgacc
       61 tgttcgttgc aacaaattga tgagcaatgc ttttttataa tgccaacttt gtatacaaaa
      121 gtaggctgga tgaatcgaat aacttcttct tcaaatgcgc aaacgaagaa atttcctcag
      181 tctcagccac agccgccgga gaaaaatgag aaaaaactga aaagggagag atttccggtg
      241 aaccctttga gagatttcag tacaagaagc tctggaagca gcagttgtag caatgagagt
      301 gcttctggtt caacttccgg tgaagcatcc aacggttgtc acaggttctt gctctctcat
      361 tctttctcat cgtcttcttc atcatcttct tcttcgttgg gagtttttcc tcgtagacca
      421 gtgagatcag tagctaaaaa ccccaaatct gctccggtag ttagtaaacc cttaattaga
      481 aagaaaccaa gctctcttga ggaggttaag ttgaaatcta ctcttactga gaaacctaat
      541 ttgctgaagt ctcagagatg caagacaaat cctgtatctg gaaagagacc aacttgtaag
      601 attaccatga aaccggagaa agtttcggtt ttgaagaaac aatcttctgt ttctaggtaa
      661 gctgcagtgt tttcatcaca cgtaatatca tccattttat attatgatcc acaaacagca
      721 aaggcccatt aatctcatta tattttagat taattgattg caattgccca acttctccac
      781 tcagcaatgt tagttttaaa gacacatcaa gttagtgtat tttattttta atctttctat
      841 aaaaaacaac actttgtttt tgtctatttc ttattctgtg aaaaagagaa atgaaaaagt
      901 aaaaaagata acaaaaaggc tggtttttca cggtgagaat ttttcatggt cacgcaataa
      961 cttaaatatg aactattaaa ataactaaga ttgaaaaatt agcatgatca aaagaagaag
     1021 aaaaaaaaga caacatgaag cggaaaggaa aaaaaagaaa gctgaaaatc taagcttatc
     1081 atgagcggag aattaaggga gtcacgttat gacccccgcc gatgacgcgg gacaagccgt
     1141 tttacgtttg gaactgacag aaccgcaacg ttgaaggagc cactcagccg cgggtttctg
     1201 gagtttaatg agctaagcac atacgtcaga aaccattatt gcgcgttcaa aagtcgccta
     1261 aggtcactat cagctagcaa atatttcttg tcaaaaatgc tccactgacg ttccataaat
     1321 tcccctcggt atccaattag agtctcatat tcactctcaa tccaaataat ctgcaccgaa
     1381 tctagagtta tcaacaagtt tgtacaaaaa agctgaacga gaaacgtaaa atgatataaa
     1441 tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca
     1501 acatatccag tcactatggc ggcccctcga ggggccgcat taggcacccc aggctttaca
     1561 ctttatgctt ccggctcgta taatgtgtgg attttgagtt aggatccggc gagattttca
     1621 ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc
     1681 caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac
     1741 cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag
     1801 ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt
     1861 atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt
     1921 ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg
     1981 cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc
     2041 cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc
     2101 agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc
     2161 aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc
     2221 gtctgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag
     2281 tggcagggcg gggcgtaaag atctggatcc ggcttactaa aagccagata acagtatgcg
     2341 tatttgcgcg ctgatttttg cggtataaga atatatactg atatgtatac ccgaagtatg
     2401 tcaaaaagag gtgtgctatg aagcagcgta ttacagtgac agttgacagc gacagctatc
     2461 agttgctcaa ggcatatatg atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa
     2521 tgaagcccgt cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag ggatggctga
     2581 ggtcgcccgg tttattgaaa tgaacggctc ttttgctgac gagaacaggg actggtgaaa
     2641 tgcagtttaa ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac
     2701 agagtgatat tattgacacg cccgggcgac ggatggtgat ccccctggcc agtgcacgtc
     2761 tgctgtcaga taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct
     2821 ggcgcatgat gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg
     2881 ctgatctcag ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa
     2941 tataaatgtc aggctccctt atacacagcc agtctgcagg tcgaccatag tgactggata
     3001 tgttgtgttt tacagtatta tgtagtctgt tttttatgca aaatctaatt taatatattg
     3061 atatttatat cattttacgt ttctcgttca gctttcttgt acaaagtggt tgataacagc
     3121 atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt
     3181 ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa
     3241 tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat
     3301 ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac
     3361 atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg
     3421 gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt
     3481 gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa
     3541 acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat
     3601 gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa
     3661 aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca
     3721 tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat
     3781 ctggttccgc gtggatccgc ttagagctcg aatttccccg atcgttcaaa catttggcaa
     3841 taaagtttct taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg
     3901 ttgaattacg ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgg
     3961 gtttttatga ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag
     4021 cgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcgggaattc
     4081 tagcttcgac aattattttg gactctcatg ttctcaagct gaaatccaaa agaaacgtga
     4141 tgttggttca gatttggaat cactgacaat gaagtttcag tcagccggtt tgtcaccaag
     4201 gatccaggct tcatcatggg agccgagccg ttcaagtttc aacttcgact atttggatcc
     4261 gtcctagtgg atcccccatc acaactttgt atagaaaagt tgaacgagaa acgtaaaatg
     4321 atataaatat caatatatta aattagattt tgcataaaaa acagactaca taatactgta
     4381 aaacacaaca tatccagtca ctatggcggc cgcattaggc accccaggct ttacacttta
     4441 tgcttccggc tcgtataatg tgtggatttt gagttaggat ccggcgagat tttcaggagc
     4501 taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg
     4561 gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac
     4621 cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta
     4681 tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat tgcgatcgca
     4741 tttaaatggc cggcccttaa ttaaggcgcg ccgtttaagg atcttcacct agatcctttt
     4801 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     4861 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     4921 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     4981 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5041 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5101 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5161 cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5221 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5281 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5341 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5401 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5461 ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct
     5521 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     5581 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     5641 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     5701 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     5761 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     5821 tccgcgcaca tttccccgaa aagtgccacc tgaaacgggc gacggatggt gatccccctg
     5881 gccagtgcac gtctgctgtc agataaagtc tcccgtgaac tttacccggt ggtgcatatc
     5941 ggggatgaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc
     6001 ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg
     6061 atgttctggg gaatataaat gtcaggctcc cttatacaca gccagtctgc aggtcgatcg
     6121 accatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa
     6181 tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcaact ttattataca
     6241 aagttgtgat ccacatcttc tgattctatt gatctctcac agtttcagag aggtttagtt
     6301 gacaggagtt cttgtcatcc tcatgtaaca ctggataaag tgtcaaggac acatttgaga
     6361 gtagagcaaa caaatagcca catgcctgaa atgaaatccc aacagattac agatactgaa
     6421 tttgatattc aaaatcacaa agaaagtgct gcttctctgg gaaaaggaag agaattgctt
     6481 ccatgctctg ctgcagagtc aataagcact gatggaggtg gtctgatttg ttctgaggat
     6541 tcaaattgga tggcatgtta caagaattac ccaacttttt aatacaaagt tggcattata
     6601 agaaagcatt gcttatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc
     6661 attatttgcc atccagctgc agctctggcc cgtgtctcaa aatctctgat gttacattgc
     6721 acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac
     6781 aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat
     6841 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     6901 aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     6961 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     7021 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac
     7081 tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     7141 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     7201 tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg
     7261 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     7321 gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt
     7381 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     7441 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     7501 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     7561 taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt
     7621 gtaacactgg cagagcatta cgctgacttg acgggacggc gcaagctcat gaccaaaatc
     7681 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     7741 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     7801 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     7861 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     7921 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     7981 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     8041 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     8101 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     8161 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     8221 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     8281 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     8341 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     8401 gcgttatccc ctgattctgt ggataaccgt attaccgcta gccaggaaga gtttgtagaa
     8461 acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg
     8521 gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg
     8581 gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc
     8641 agtcttccga ctgagccttt cgttttattt gatgcctggc agttccctac tctcgc
//
