LOCUS       40924_36538        8450 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-43 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8450)
  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  JOURNAL   Unpublished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 8450)
  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 8450)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8450)
  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            "Unpublished"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..8450
                     /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    1109..1233
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1270..1300
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1354..2010
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             2355..2657
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(2701..2825)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2836..3552
                     /codon_start=1
                     /label=ECFP
                     /note="enhanced CFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     terminator      3574..3826
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4035..4159
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4184..4214
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4486..4526
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4618..5475)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5476..5580)
                     /label=AmpR promoter
     protein_bind    complement(5877..6000)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6326..6421)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6545..7351
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7497..8085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(8220..8247)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8339..8425)
                     /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 tcgactctag agttatcaac aagtttgtac aaaaaagctg aacgagaaac
     1141 gtaaaatgat ataaatatca atatattaaa ttagattttg cataaaaaac agactacata
     1201 atactgtaaa acacaacata tccagtcact atggcggccc ctcgaggggc cgcattaggc
     1261 accccaggct ttacacttta tgcttccggc tcgtataatg tgtggatttt gagttaggat
     1321 ccggcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc
     1381 accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct
     1441 caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag
     1501 aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct
     1561 catccggaat tccgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac
     1621 ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac
     1681 cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa
     1741 aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc
     1801 tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc
     1861 gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt
     1921 caggttcatc atgccgtctg tgatggcttc catgtcggca gaatgcttaa tgaattacaa
     1981 cagtactgcg atgagtggca gggcggggcg taaagatctg gatccggctt actaaaagcc
     2041 agataacagt atgcgtattt gcgcgctgat ttttgcggta taagaatata tactgatatg
     2101 tatacccgaa gtatgtcaaa aagaggtgtg ctatgaagca gcgtattaca gtgacagttg
     2161 acagcgacag ctatcagttg ctcaaggcat atatgatgtc aatatctccg gtctggtaag
     2221 cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca
     2281 ggaagggatg gctgaggtcg cccggtttat tgaaatgaac ggctcttttg ctgacgagaa
     2341 cagggactgg tgaaatgcag tttaaggttt acacctataa aagagagagc cgttatcgtc
     2401 tgtttgtgga tgtacagagt gatattattg acacgcccgg gcgacggatg gtgatccccc
     2461 tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata
     2521 tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtctccgtta
     2581 tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc
     2641 tgatgttctg gggaatataa atgtcaggct cccttataca cagccagtct gcaggtcgac
     2701 catagtgact ggatatgttg tgttttacag tattatgtag tctgtttttt atgcaaaatc
     2761 taatttaata tattgatatt tatatcattt tacgtttctc gttcagcttt cttgtacaaa
     2821 gtggttgata acagcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     2881 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     2941 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     3001 gtgccctggc ccaccctcgt gaccaccctg acctggggcg tgcagtgctt cagccgctac
     3061 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     3121 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     3181 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     3241 aacatcctgg ggcacaagct ggagtacaac tacatcagcc acaacgtcta tatcaccgcc
     3301 gacaagcaga agaacggcat caaggccaac ttcaagatcc gccacaacat cgaggacggc
     3361 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     3421 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     3481 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     3541 gagctgtaca aggcttagag ctcgaatttc cccgatcgtt caaacatttg gcaataaagt
     3601 ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat
     3661 tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt
     3721 atgattagag tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca
     3781 aactaggata aattatcgcg cgcggtgtca tctatgttac tagatcggga attctagctt
     3841 cgacaattat tttggactct catgttctca agctgaaatc caaaagaaac gtgatgttgg
     3901 ttcagatttg gaatcactga caatgaagtt tcagtcagcc ggtttgtcac caaggatcca
     3961 ggcttcatca tgggagccga gccgttcaag tttcaacttc gactatttgg atccgtccta
     4021 gtggatcccc catcacaact ttgtatagaa aagttgaacg agaaacgtaa aatgatataa
     4081 atatcaatat attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac
     4141 aacatatcca gtcactatgg cggccgcatt aggcacccca ggctttacac tttatgcttc
     4201 cggctcgtat aatgtgtgga ttttgagtta ggatccggcg agattttcag gagctaagga
     4261 agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg
     4321 taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca
     4381 gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc
     4441 ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattgcgat cgcatttaaa
     4501 tggccggccc ttaattaagg cgcgccgttt aaggatcttc acctagatcc ttttaaatta
     4561 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4621 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4681 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4741 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4801 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4861 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     4921 tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     4981 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5041 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5101 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5161 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5221 cccggcgtca acacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5281 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5341 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5401 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5461 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5521 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5581 cacatttccc cgaaaagtgc cacctgaaac gggcgacgga tggtgatccc cctggccagt
     5641 gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat
     5701 gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa
     5761 gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc
     5821 tggggaatat aaatgtcagg ctcccttata cacagccagt ctgcaggtcg atcgaccata
     5881 gtgactggat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc aaaatctaat
     5941 ttaatatatt gatatttata tcattttacg tttctcgttc aactttatta tacaaagttg
     6001 tgatccacat cttctgattc tattgatctc tcacagtttc agagaggttt agttgacagg
     6061 agttcttgtc atcctcatgt aacactggat aaagtgtcaa ggacacattt gagagtagag
     6121 caaacaaata gccacatgcc tgaaatgaaa tcccaacaga ttacagatac tgaatttgat
     6181 attcaaaatc acaaagaaag tgctgcttct ctgggaaaag gaagagaatt gcttccatgc
     6241 tctgctgcag agtcaataag cactgatgga ggtggtctga tttgttctga ggattcaaat
     6301 tggatggcat gttacaagaa ttacccaact ttttaataca aagttggcat tataagaaag
     6361 cattgcttat caatttgttg caacgaacag gtcactatca gtcaaaataa aatcattatt
     6421 tgccatccag ctgcagctct ggcccgtgtc tcaaaatctc tgatgttaca ttgcacaaga
     6481 taaaaatata tcatcatgaa caataaaact gtctgcttac ataaacagta atacaagggg
     6541 tgttatgagc catattcaac gggaaacgtc gaggccgcga ttaaattcca acatggatgc
     6601 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     6661 tcgcttgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     6721 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     6781 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     6841 ccccggaaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     6901 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     6961 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     7021 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     7081 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     7141 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     7201 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     7261 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     7321 gcagtttcat ttgatgctcg atgagttttt ctaatcagaa ttggttaatt ggttgtaaca
     7381 ctggcagagc attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa
     7441 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     7501 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     7561 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     7621 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     7681 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     7741 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     7801 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     7861 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     7921 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     7981 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     8041 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     8101 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     8161 tcccctgatt ctgtggataa ccgtattacc gctagccagg aagagtttgt agaaacgcaa
     8221 aaaggccatc cgtcaggatg gccttctgct tagtttgatg cctggcagtt tatggcgggc
     8281 gtcctgcccg ccaccctccg ggccgttgct tcacaacgtt caaatccgct cccggcggat
     8341 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
     8401 ccgactgagc ctttcgtttt atttgatgcc tggcagttcc ctactctcgc
//
