LOCUS       40924_36633        8420 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-20 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8420)
  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  JOURNAL   Unpublished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 8420)
  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 8420)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8420)
  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            "Unpublished"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..8420
                     /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             3136..3165
                     /codon_start=1
                     /label=Myc
                     /note="Myc (human c-Myc proto-oncogene) epitope tag"
                     /translation="EQKLISEEDL"
     CDS             3172..3201
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3217..3246
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3253..3282
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3289..3318
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3334..3363
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3370..3399
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3406..3435
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3451..3480
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3487..3516
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     terminator      3544..3796
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4005..4129
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4154..4184
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4456..4496
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4588..5445)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5446..5550)
                     /label=AmpR promoter
     protein_bind    complement(5847..5970)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6296..6391)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6515..7321
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7467..8055
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(8190..8217)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8309..8395)
                     /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 gggttaatta acggtgaaca aaagctaatc tccgaggaag acttgaacgg tgaacaaaaa
     3181 ttaatctcag aagaagactt gaacggactc gacggtgaac aaaagttgat ttctgaagaa
     3241 gatttgaacg gtgaacaaaa gctaatctcc gaggaagact tgaacggtga acaaaaatta
     3301 atctcagaag aagacttgaa cggactcgac ggtgaacaaa agttgatttc tgaagaagat
     3361 ttgaacggtg aacaaaagct aatctccgag gaagacttga acggtgaaca aaaattaatc
     3421 tcagaagaag acttgaacgg actcgacggt gaacaaaagt tgatttctga agaagatttg
     3481 aacggtgaac aaaagctaat ctccgaggaa gacttgaacg gtgcttagag ctcgaatttc
     3541 cccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt
     3601 gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa
     3661 tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa
     3721 tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca
     3781 tctatgttac tagatcggga attctagctt cgacaattat tttggactct catgttctca
     3841 agctgaaatc caaaagaaac gtgatgttgg ttcagatttg gaatcactga caatgaagtt
     3901 tcagtcagcc ggtttgtcac caaggatcca ggcttcatca tgggagccga gccgttcaag
     3961 tttcaacttc gactatttgg atccgtccta gtggatcccc catcacaact ttgtatagaa
     4021 aagttgaacg agaaacgtaa aatgatataa atatcaatat attaaattag attttgcata
     4081 aaaaacagac tacataatac tgtaaaacac aacatatcca gtcactatgg cggccgcatt
     4141 aggcacccca ggctttacac tttatgcttc cggctcgtat aatgtgtgga ttttgagtta
     4201 ggatccggcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata
     4261 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
     4321 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt
     4381 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa
     4441 tgctcatccg gaattgcgat cgcatttaaa tggccggccc ttaattaagg cgcgccgttt
     4501 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     4561 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     4621 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     4681 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     4741 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     4801 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     4861 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg
     4921 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     4981 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     5041 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     5101 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     5161 atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc
     5221 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     5281 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     5341 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     5401 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     5461 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     5521 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgaaac
     5581 gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt
     5641 gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg
     5701 gccagtgtgc cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat
     5761 gacatcaaaa acgccattaa cctgatgttc tggggaatat aaatgtcagg ctcccttata
     5821 cacagccagt ctgcaggtcg atcgaccata gtgactggat atgttgtgtt ttacagtatt
     5881 atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg
     5941 tttctcgttc aactttatta tacaaagttg tgatccacat cttctgattc tattgatctc
     6001 tcacagtttc agagaggttt agttgacagg agttcttgtc atcctcatgt aacactggat
     6061 aaagtgtcaa ggacacattt gagagtagag caaacaaata gccacatgcc tgaaatgaaa
     6121 tcccaacaga ttacagatac tgaatttgat attcaaaatc acaaagaaag tgctgcttct
     6181 ctgggaaaag gaagagaatt gcttccatgc tctgctgcag agtcaataag cactgatgga
     6241 ggtggtctga tttgttctga ggattcaaat tggatggcat gttacaagaa ttacccaact
     6301 ttttaataca aagttggcat tataagaaag cattgcttat caatttgttg caacgaacag
     6361 gtcactatca gtcaaaataa aatcattatt tgccatccag ctgcagctct ggcccgtgtc
     6421 tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact
     6481 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
     6541 gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     6601 taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga
     6661 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     6721 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
     6781 tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcattcc aggtattaga
     6841 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     6901 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca
     6961 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     7021 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     7081 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     7141 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     7201 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     7261 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt
     7321 ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc attacgctga cttgacggga
     7381 cggcgcaagc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     7441 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     7501 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     7561 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     7621 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     7681 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     7741 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     7801 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     7861 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     7921 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7981 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     8041 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     8101 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     8161 gctagccagg aagagtttgt agaaacgcaa aaaggccatc cgtcaggatg gccttctgct
     8221 tagtttgatg cctggcagtt tatggcgggc gtcctgcccg ccaccctccg ggccgttgct
     8281 tcacaacgtt caaatccgct cccggcggat ttgtcctact caggagagcg ttcaccgaca
     8341 aacaacagat aaaacgaaag gcccagtctt ccgactgagc ctttcgtttt atttgatgcc
     8401 tggcagttcc ctactctcgc
//
