LOCUS       40924_36513        8396 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-22 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8396)
  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  JOURNAL   Unpublished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 8396)
  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 8396)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8396)
  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            "Unpublished"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..8396
                     /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"
     protein_bind    1094..1218
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1255..1285
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1339..1995
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             2340..2642
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(2686..2810)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2821..3474
                     /codon_start=1
                     /label=GST
                     /note="glutathione S-transferase from Schistosoma
                     japonicum"
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             3481..3498
                     /codon_start=1
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
                     /translation="LVPRGS"
     terminator      3520..3772
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    3981..4105
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4130..4160
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4432..4472
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4564..5421)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5422..5526)
                     /label=AmpR promoter
     protein_bind    complement(5823..5946)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6272..6367)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6491..7297
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7443..8031
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(8166..8193)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8285..8371)
                     /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 taagcttggg
     1081 tctagagtta tcaacaagtt tgtacaaaaa agctgaacga gaaacgtaaa atgatataaa
     1141 tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca
     1201 acatatccag tcactatggc ggcccctcga ggggccgcat taggcacccc aggctttaca
     1261 ctttatgctt ccggctcgta taatgtgtgg attttgagtt aggatccggc gagattttca
     1321 ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc
     1381 caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac
     1441 cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag
     1501 ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt
     1561 atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt
     1621 ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg
     1681 cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc
     1741 cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc
     1801 agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc
     1861 aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc
     1921 gtctgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag
     1981 tggcagggcg gggcgtaaag atctggatcc ggcttactaa aagccagata acagtatgcg
     2041 tatttgcgcg ctgatttttg cggtataaga atatatactg atatgtatac ccgaagtatg
     2101 tcaaaaagag gtgtgctatg aagcagcgta ttacagtgac agttgacagc gacagctatc
     2161 agttgctcaa ggcatatatg atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa
     2221 tgaagcccgt cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag ggatggctga
     2281 ggtcgcccgg tttattgaaa tgaacggctc ttttgctgac gagaacaggg actggtgaaa
     2341 tgcagtttaa ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac
     2401 agagtgatat tattgacacg cccgggcgac ggatggtgat ccccctggcc agtgcacgtc
     2461 tgctgtcaga taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct
     2521 ggcgcatgat gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg
     2581 ctgatctcag ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa
     2641 tataaatgtc aggctccctt atacacagcc agtctgcagg tcgaccatag tgactggata
     2701 tgttgtgttt tacagtatta tgtagtctgt tttttatgca aaatctaatt taatatattg
     2761 atatttatat cattttacgt ttctcgttca gctttcttgt acaaagtggt tgataacagc
     2821 atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt
     2881 ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa
     2941 tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat
     3001 ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac
     3061 atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg
     3121 gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt
     3181 gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa
     3241 acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat
     3301 gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa
     3361 aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca
     3421 tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat
     3481 ctggttccgc gtggatccgc ttagagctcg aatttccccg atcgttcaaa catttggcaa
     3541 taaagtttct taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg
     3601 ttgaattacg ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgg
     3661 gtttttatga ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag
     3721 cgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcgggaattc
     3781 tagcttcgac aattattttg gactctcatg ttctcaagct gaaatccaaa agaaacgtga
     3841 tgttggttca gatttggaat cactgacaat gaagtttcag tcagccggtt tgtcaccaag
     3901 gatccaggct tcatcatggg agccgagccg ttcaagtttc aacttcgact atttggatcc
     3961 gtcctagtgg atcccccatc acaactttgt atagaaaagt tgaacgagaa acgtaaaatg
     4021 atataaatat caatatatta aattagattt tgcataaaaa acagactaca taatactgta
     4081 aaacacaaca tatccagtca ctatggcggc cgcattaggc accccaggct ttacacttta
     4141 tgcttccggc tcgtataatg tgtggatttt gagttaggat ccggcgagat tttcaggagc
     4201 taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg
     4261 gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac
     4321 cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta
     4381 tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat tgcgatcgca
     4441 tttaaatggc cggcccttaa ttaaggcgcg ccgtttaagg atcttcacct agatcctttt
     4501 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     4561 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     4621 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     4681 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     4741 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     4801 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     4861 cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     4921 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     4981 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5041 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5101 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     5161 ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct
     5221 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     5281 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     5341 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     5401 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     5461 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     5521 tccgcgcaca tttccccgaa aagtgccacc tgaaacgggc gacggatggt gatccccctg
     5581 gccagtgcac gtctgctgtc agataaagtc tcccgtgaac tttacccggt ggtgcatatc
     5641 ggggatgaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc
     5701 ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg
     5761 atgttctggg gaatataaat gtcaggctcc cttatacaca gccagtctgc aggtcgatcg
     5821 accatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa
     5881 tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcaact ttattataca
     5941 aagttgtgat ccacatcttc tgattctatt gatctctcac agtttcagag aggtttagtt
     6001 gacaggagtt cttgtcatcc tcatgtaaca ctggataaag tgtcaaggac acatttgaga
     6061 gtagagcaaa caaatagcca catgcctgaa atgaaatccc aacagattac agatactgaa
     6121 tttgatattc aaaatcacaa agaaagtgct gcttctctgg gaaaaggaag agaattgctt
     6181 ccatgctctg ctgcagagtc aataagcact gatggaggtg gtctgatttg ttctgaggat
     6241 tcaaattgga tggcatgtta caagaattac ccaacttttt aatacaaagt tggcattata
     6301 agaaagcatt gcttatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc
     6361 attatttgcc atccagctgc agctctggcc cgtgtctcaa aatctctgat gttacattgc
     6421 acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac
     6481 aaggggtgtt atgagccata ttcaacggga aacgtcgagg ccgcgattaa attccaacat
     6541 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     6601 aatctatcgc ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     6661 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     6721 gcctcttccg accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac
     6781 tgcgatcccc ggaaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     6841 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     6901 tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg
     6961 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     7021 gaaagaaatg cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt
     7081 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     7141 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     7201 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     7261 taaattgcag tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt
     7321 gtaacactgg cagagcatta cgctgacttg acgggacggc gcaagctcat gaccaaaatc
     7381 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     7441 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     7501 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     7561 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     7621 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     7681 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     7741 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     7801 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     7861 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     7921 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     7981 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     8041 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     8101 gcgttatccc ctgattctgt ggataaccgt attaccgcta gccaggaaga gtttgtagaa
     8161 acgcaaaaag gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg
     8221 gcgggcgtcc tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg
     8281 gcggatttgt cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc
     8341 agtcttccga ctgagccttt cgttttattt gatgcctggc agttccctac tctcgc
//
