LOCUS       40924_36518        7751 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-25 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7751)
  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  JOURNAL   Unpublished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 7751)
  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 7751)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7751)
  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            "Unpublished"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..7751
                     /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..2853
                     /codon_start=1
                     /label=T7 tag (gene 10 leader)
                     /note="leader peptide from bacteriophage T7 gene 10"
                     /translation="MASMTGGQQMG"
     terminator      2875..3127
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    3336..3460
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3485..3515
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    3787..3827
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(3919..4776)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4777..4881)
                     /label=AmpR promoter
     protein_bind    complement(5178..5301)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(5627..5722)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             5846..6652
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      6798..7386
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(7521..7548)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(7640..7726)
                     /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 atggctagca tgactggtgg acagcaaatg ggtgcttaga gctcgaattt ccccgatcgt
     2881 tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt
     2941 atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta atgcatgacg
     3001 ttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta atacgcgata
     3061 gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc atctatgtta
     3121 ctagatcggg aattctagct tcgacaatta ttttggactc tcatgttctc aagctgaaat
     3181 ccaaaagaaa cgtgatgttg gttcagattt ggaatcactg acaatgaagt ttcagtcagc
     3241 cggtttgtca ccaaggatcc aggcttcatc atgggagccg agccgttcaa gtttcaactt
     3301 cgactatttg gatccgtcct agtggatccc ccatcacaac tttgtataga aaagttgaac
     3361 gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga
     3421 ctacataata ctgtaaaaca caacatatcc agtcactatg gcggccgcat taggcacccc
     3481 aggctttaca ctttatgctt ccggctcgta taatgtgtgg attttgagtt aggatccggc
     3541 gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt
     3601 tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg
     3661 tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa
     3721 taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc
     3781 ggaattgcga tcgcatttaa atggccggcc cttaattaag gcgcgccgtt taaggatctt
     3841 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3901 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3961 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4021 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     4081 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4141 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     4201 taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt
     4261 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4321 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4381 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4441 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4501 gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag
     4561 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4621 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4681 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4741 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4801 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4861 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgaaa cgggcgacgg
     4921 atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac
     4981 ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg
     5041 ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa
     5101 aacgccatta acctgatgtt ctggggaata taaatgtcag gctcccttat acacagccag
     5161 tctgcaggtc gatcgaccat agtgactgga tatgttgtgt tttacagtat tatgtagtct
     5221 gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt
     5281 caactttatt atacaaagtt gtgatccaca tcttctgatt ctattgatct ctcacagttt
     5341 cagagaggtt tagttgacag gagttcttgt catcctcatg taacactgga taaagtgtca
     5401 aggacacatt tgagagtaga gcaaacaaat agccacatgc ctgaaatgaa atcccaacag
     5461 attacagata ctgaatttga tattcaaaat cacaaagaaa gtgctgcttc tctgggaaaa
     5521 ggaagagaat tgcttccatg ctctgctgca gagtcaataa gcactgatgg aggtggtctg
     5581 atttgttctg aggattcaaa ttggatggca tgttacaaga attacccaac tttttaatac
     5641 aaagttggca ttataagaaa gcattgctta tcaatttgtt gcaacgaaca ggtcactatc
     5701 agtcaaaata aaatcattat ttgccatcca gctgcagctc tggcccgtgt ctcaaaatct
     5761 ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac tgtctgctta
     5821 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cgaggccgcg
     5881 attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg
     5941 gcaatcaggt gcgacaatct atcgcttgta tgggaagccc gatgcgccag agttgtttct
     6001 gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca gactaaactg
     6061 gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc
     6121 atggttactc accactgcga tccccggaaa aacagcattc caggtattag aagaatatcc
     6181 tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat
     6241 tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc
     6301 acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta atggctggcc
     6361 tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg attcagtcgt
     6421 cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg
     6481 tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa
     6541 ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga
     6601 taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt tctaatcaga
     6661 attggttaat tggttgtaac actggcagag cattacgctg acttgacggg acggcgcaag
     6721 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6781 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6841 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6901 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     6961 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     7021 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     7081 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     7141 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     7201 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     7261 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     7321 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     7381 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     7441 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgctagccag
     7501 gaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttagtttgat
     7561 gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt
     7621 tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga
     7681 taaaacgaaa ggcccagtct tccgactgag cctttcgttt tatttgatgc ctggcagttc
     7741 cctactctcg c
//
