LOCUS       40924_36648        8698 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-24 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8698)
  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  JOURNAL   Unpublished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 8698)
  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 8698)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8698)
  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            "Unpublished"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..8698
                     /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"
     CDS             1393..2046
                     /codon_start=1
                     /label=GST
                     /note="glutathione S-transferase from Schistosoma
                     japonicum"
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             2053..2070
                     /codon_start=1
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
     protein_bind    2080..2204
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2241..2271
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             2325..2981
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             3326..3628
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3672..3796)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      3822..4074
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4283..4407
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4432..4462
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4734..4774
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4866..5723)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5724..5828)
                     /label=AmpR promoter
     protein_bind    complement(6125..6248)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6574..6669)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6793..7599
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7745..8333
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(8468..8495)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8587..8673)
                     /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 tctagaatga gcatgtcccc tatactaggt tattggaaaa ttaagggcct tgtgcaaccc
     1441 actcgacttc ttttggaata tcttgaagaa aaatatgaag agcatttgta tgagcgcgat
     1501 gaaggtgata aatggcgaaa caaaaagttt gaattgggtt tggagtttcc caatcttcct
     1561 tattatattg atggtgatgt taaattaaca cagtctatgg ccatcatacg ttatatagct
     1621 gacaagcaca acatgttggg tggttgtcca aaagagcgtg cagagatttc aatgcttgaa
     1681 ggagcggttt tggatattag atacggtgtt tcgagaattg catatagtaa agactttgaa
     1741 actctcaaag ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt cgaagatcgt
     1801 ttatgtcata aaacatattt aaatggtgat catgtaaccc atcctgactt catgttgtat
     1861 gacgctcttg atgttgtttt atacatggac ccaatgtgcc tggatgcgtt cccaaaatta
     1921 gtttgtttta aaaaacgtat tgaagctatc ccacaaattg ataagtactt gaaatccagc
     1981 aagtatatag catggccttt gcagggctgg caagccacgt ttggtggtgg cgaccatcct
     2041 ccaaaatcgg atctggttcc gcgtggatcc gctgttatca caagtttgta caaaaaagct
     2101 gaacgagaaa cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa
     2161 cagactacat aatactgtaa aacacaacat atccagtcac tatggcggcc cctcgagggg
     2221 ccgcattagg caccccaggc tttacacttt atgcttccgg ctcgtataat gtgtggattt
     2281 tgagttagga tccggcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca
     2341 ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc
     2401 agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa
     2461 agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc
     2521 tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg
     2581 atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct
     2641 ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt
     2701 gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct
     2761 cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact
     2821 tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc
     2881 cgctggcgat tcaggttcat catgccgtct gtgatggctt ccatgtcggc agaatgctta
     2941 atgaattaca acagtactgc gatgagtggc agggcggggc gtaaacgcgt ggatccggct
     3001 tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat
     3061 atactgatat gtatacccga agtatgtcaa aaagaggtgt gctatgaagc agcgtattac
     3121 agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt caatatctcc
     3181 ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa
     3241 gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa cggctctttt
     3301 gctgacgaga acagggactg gtgaaatgca gtttaaggtt tacacctata aaagagagag
     3361 ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat
     3421 ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc
     3481 ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc
     3541 ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa
     3601 cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac acagccagtc
     3661 tgcaggtcga ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt
     3721 tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcagctt
     3781 tcttgtacaa agtggtgata acctagagct cgaatttccc cgatcgttca aacatttggc
     3841 aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc
     3901 tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat
     3961 gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat
     4021 agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat
     4081 tctagcttcg acaattattt tggactctca tgttctcaag ctgaaatcca aaagaaacgt
     4141 gatgttggtt cagatttgga atcactgaca atgaagtttc agtcagccgg tttgtcacca
     4201 aggatccagg cttcatcatg ggagccgagc cgttcaagtt tcaacttcga ctatttggat
     4261 ccgtcctagt ggatccccca tcacaacttt gtatagaaaa gttgaacgag aaacgtaaaa
     4321 tgatataaat atcaatatat taaattagat tttgcataaa aaacagacta cataatactg
     4381 taaaacacaa catatccagt cactatggcg gccgcattag gcaccccagg ctttacactt
     4441 tatgcttccg gctcgtataa tgtgtggatt ttgagttagg atccggcgag attttcagga
     4501 gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa
     4561 tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag
     4621 accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt
     4681 tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attgcgatcg
     4741 catttaaatg gccggccctt aattaaggcg cgccgtttaa ggatcttcac ctagatcctt
     4801 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4861 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4921 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4981 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5041 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5101 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5161 aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5221 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5281 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5341 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5401 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5461 tgctcttgcc cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg
     5521 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5581 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5641 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5701 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5761 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5821 gttccgcgca catttccccg aaaagtgcca cctgaaacgg gcgacggatg gtgatccccc
     5881 tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata
     5941 tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtctccgtta
     6001 tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc
     6061 tgatgttctg gggaatataa atgtcaggct cccttataca cagccagtct gcaggtcgat
     6121 cgaccatagt gactggatat gttgtgtttt acagtattat gtagtctgtt ttttatgcaa
     6181 aatctaattt aatatattga tatttatatc attttacgtt tctcgttcaa ctttattata
     6241 caaagttgtg atccacatct tctgattcta ttgatctctc acagtttcag agaggtttag
     6301 ttgacaggag ttcttgtcat cctcatgtaa cactggataa agtgtcaagg acacatttga
     6361 gagtagagca aacaaatagc cacatgcctg aaatgaaatc ccaacagatt acagatactg
     6421 aatttgatat tcaaaatcac aaagaaagtg ctgcttctct gggaaaagga agagaattgc
     6481 ttccatgctc tgctgcagag tcaataagca ctgatggagg tggtctgatt tgttctgagg
     6541 attcaaattg gatggcatgt tacaagaatt acccaacttt ttaatacaaa gttggcatta
     6601 taagaaagca ttgcttatca atttgttgca acgaacaggt cactatcagt caaaataaaa
     6661 tcattatttg ccatccagct gcagctctgg cccgtgtctc aaaatctctg atgttacatt
     6721 gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat aaacagtaat
     6781 acaaggggtg ttatgagcca tattcaacgg gaaacgtcga ggccgcgatt aaattccaac
     6841 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     6901 acaatctatc gcttgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     6961 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     7021 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
     7081 actgcgatcc ccggaaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     7141 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     7201 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
     7261 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     7321 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     7381 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     7441 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     7501 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     7561 aataaattgc agtttcattt gatgctcgat gagtttttct aatcagaatt ggttaattgg
     7621 ttgtaacact ggcagagcat tacgctgact tgacgggacg gcgcaagctc atgaccaaaa
     7681 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     7741 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     7801 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     7861 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     7921 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     7981 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     8041 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     8101 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     8161 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     8221 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     8281 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     8341 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     8401 ctgcgttatc ccctgattct gtggataacc gtattaccgc tagccaggaa gagtttgtag
     8461 aaacgcaaaa aggccatccg tcaggatggc cttctgctta gtttgatgcc tggcagttta
     8521 tggcgggcgt cctgcccgcc accctccggg ccgttgcttc acaacgttca aatccgctcc
     8581 cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc
     8641 ccagtcttcc gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgc
//
