LOCUS       40924_36483        8440 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-04 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8440)
  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  JOURNAL   Unpublished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 8440)
  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 8440)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8440)
  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            "Unpublished"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..8440
                     /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    1099..1223
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1260..1290
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1344..2000
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             2345..2647
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(2691..2815)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2820..3536
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     terminator      3564..3816
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4025..4149
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4174..4204
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4476..4516
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4608..5465)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5466..5570)
                     /label=AmpR promoter
     protein_bind    complement(5867..5990)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6316..6411)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6535..7341
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7487..8075
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(8210..8237)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8329..8415)
                     /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 taagcttgca
     1081 tgcctgcagg ctctaatcac aagtttgtac aaaaaagctg aacgagaaac gtaaaatgat
     1141 ataaatatca atatattaaa ttagattttg cataaaaaac agactacata atactgtaaa
     1201 acacaacata tccagtcact atggcggccc ctcgaggggc cgcattaggc accccaggct
     1261 ttacacttta tgcttccggc tcgtataatg tgtggatttt gagttaggat ccggcgagat
     1321 tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata
     1381 tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct
     1441 ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc
     1501 acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat
     1561 tccgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca
     1621 ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt
     1681 tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct
     1741 atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt
     1801 tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca
     1861 tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc
     1921 atgccgtctg tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg
     1981 atgagtggca gggcggggcg taaacgcgtg gatccggctt actaaaagcc agataacagt
     2041 atgcgtattt gcgcgctgat ttttgcggta taagaatata tactgatatg tatacccgaa
     2101 gtatgtcaaa aagaggtgtg ctatgaagca gcgtattaca gtgacagttg acagcgacag
     2161 ctatcagttg ctcaaggcat atatgatgtc aatatctccg gtctggtaag cacaaccatg
     2221 cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca ggaagggatg
     2281 gctgaggtcg cccggtttat tgaaatgaac ggctcttttg ctgacgagaa cagggactgg
     2341 tgaaatgcag tttaaggttt acacctataa aagagagagc cgttatcgtc tgtttgtgga
     2401 tgtacagagt gatattattg acacgcccgg gcgacggatg gtgatccccc tggccagtgc
     2461 acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata tcggggatga
     2521 aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtctccgtta tcggggaaga
     2581 agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc tgatgttctg
     2641 gggaatataa atgtcaggct cccttataca cagccagtct gcaggtcgac catagtgact
     2701 ggatatgttg tgttttacag tattatgtag tctgtttttt atgcaaaatc taatttaata
     2761 tattgatatt tatatcattt tacgtttctc gttcagcttt cttgtacaaa gtggtgatca
     2821 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     2881 gcgacgtgaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     2941 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     3001 tcgtgaccac cttcacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     3061 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     3121 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     3181 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     3241 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     3301 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     3361 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     3421 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     3481 tgctggagtt cgtgaccgcc gccgggatca ctcacggcat ggacgagctg tacaagtaaa
     3541 gcggcccgag ctcgaatttc cccgatcgtt caaacatttg gcaataaagt ttcttaagat
     3601 tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc
     3661 atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag
     3721 tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata
     3781 aattatcgcg cgcggtgtca tctatgttac tagatcggga attctagctt cgacaattat
     3841 tttggactct catgttctca agctgaaatc caaaagaaac gtgatgttgg ttcagatttg
     3901 gaatcactga caatgaagtt tcagtcagcc ggtttgtcac caaggatcca ggcttcatca
     3961 tgggagccga gccgttcaag tttcaacttc gactatttgg atccgtccta gtggatcccc
     4021 catcacaact ttgtatagaa aagttgaacg agaaacgtaa aatgatataa atatcaatat
     4081 attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca
     4141 gtcactatgg cggccgcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     4201 aatgtgtgga ttttgagtta ggatccggcg agattttcag gagctaagga agctaaaatg
     4261 gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat
     4321 tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt
     4381 acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac
     4441 attcttgccc gcctgatgaa tgctcatccg gaattgcgat cgcatttaaa tggccggccc
     4501 ttaattaagg cgcgccgttt aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4561 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4621 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4681 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4741 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4801 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4861 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4921 gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4981 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5041 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5101 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5161 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5221 acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5281 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     5341 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5401 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5461 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5521 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5581 cgaaaagtgc cacctgaaac gggcgacgga tggtgatccc cctggccagt gcacgtctgc
     5641 tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc
     5701 gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg
     5761 atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat
     5821 aaatgtcagg ctcccttata cacagccagt ctgcaggtcg atcgaccata gtgactggat
     5881 atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat ttaatatatt
     5941 gatatttata tcattttacg tttctcgttc aactttatta tacaaagttg tgatccacat
     6001 cttctgattc tattgatctc tcacagtttc agagaggttt agttgacagg agttcttgtc
     6061 atcctcatgt aacactggat aaagtgtcaa ggacacattt gagagtagag caaacaaata
     6121 gccacatgcc tgaaatgaaa tcccaacaga ttacagatac tgaatttgat attcaaaatc
     6181 acaaagaaag tgctgcttct ctgggaaaag gaagagaatt gcttccatgc tctgctgcag
     6241 agtcaataag cactgatgga ggtggtctga tttgttctga ggattcaaat tggatggcat
     6301 gttacaagaa ttacccaact ttttaataca aagttggcat tataagaaag cattgcttat
     6361 caatttgttg caacgaacag gtcactatca gtcaaaataa aatcattatt tgccatccag
     6421 ctgcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata
     6481 tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     6541 catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc tgatttatat
     6601 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgcttgtat
     6661 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat
     6721 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc
     6781 aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat ccccggaaaa
     6841 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg
     6901 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat
     6961 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt
     7021 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa
     7081 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt
     7141 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac
     7201 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag
     7261 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat
     7321 ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc
     7381 attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa cgtgagtttt
     7441 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     7501 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     7561 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     7621 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     7681 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     7741 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     7801 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     7861 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     7921 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     7981 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     8041 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     8101 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     8161 ctgtggataa ccgtattacc gctagccagg aagagtttgt agaaacgcaa aaaggccatc
     8221 cgtcaggatg gccttctgct tagtttgatg cctggcagtt tatggcgggc gtcctgcccg
     8281 ccaccctccg ggccgttgct tcacaacgtt caaatccgct cccggcggat ttgtcctact
     8341 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt ccgactgagc
     8401 ctttcgtttt atttgatgcc tggcagttcc ctactctcgc
//
