LOCUS       40924_36553        8447 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-59 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8447)
  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  JOURNAL   Unpublished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 8447)
  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 8447)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8447)
  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            "Unpublished"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..8447
                     /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..3546
                     /codon_start=1
                     /label=TagRFP
                     /note="monomeric derivative of red fluorescent protein from
                     Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     terminator      3571..3823
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    4032..4156
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4181..4211
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4483..4523
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4615..5472)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5473..5577)
                     /label=AmpR promoter
     protein_bind    complement(5874..5997)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(6323..6418)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6542..7348
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7494..8082
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(8217..8244)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(8336..8422)
                     /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 gtctaagggc gaagagctga ttaaggagaa catgcacatg
     2881 aagctgtaca tggagggcac cgtgaacaac caccacttca agtgcacatc cgagggcgaa
     2941 ggcaagccct acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc
     3001 cccttcgcct tcgacatcct ggctaccagc ttcatgtacg gcagcagaac cttcatcaac
     3061 cacacccagg gcatccccga cttctttaag cagtccttcc ctgagggctt cacatgggag
     3121 agagtcacca catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag
     3181 gacggctgcc tcatctacaa cgtcaagatc agaggggtga acttcccatc caacggccct
     3241 gtgatgcaga agaaaacact cggctgggag gccaacaccg agatgctgta ccccgctgac
     3301 ggcggcctgg aaggcagaag cgacatggcc ctgaagctcg tgggcggggg ccacctgatc
     3361 tgcaacttca agaccacata cagatccaag aaacccgcta agaacctcaa gatgcccggc
     3421 gtctactatg tggaccacag actggaaaga atcaaggagg ccgacaaaga gacctacgtc
     3481 gagcagcacg aggtggctgt ggccagatac tgcgacctcc ctagcaaact ggggcacaaa
     3541 cttaattgag cttagagctc gaatttcccc gatcgttcaa acatttggca ataaagtttc
     3601 ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac
     3661 gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg
     3721 attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac
     3781 taggataaat tatcgcgcgc ggtgtcatct atgttactag atcgggaatt ctagcttcga
     3841 caattatttt ggactctcat gttctcaagc tgaaatccaa aagaaacgtg atgttggttc
     3901 agatttggaa tcactgacaa tgaagtttca gtcagccggt ttgtcaccaa ggatccaggc
     3961 ttcatcatgg gagccgagcc gttcaagttt caacttcgac tatttggatc cgtcctagtg
     4021 gatcccccat cacaactttg tatagaaaag ttgaacgaga aacgtaaaat gatataaata
     4081 tcaatatatt aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac
     4141 atatccagtc actatggcgg ccgcattagg caccccaggc tttacacttt atgcttccgg
     4201 ctcgtataat gtgtggattt tgagttagga tccggcgaga ttttcaggag ctaaggaagc
     4261 taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa
     4321 agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct
     4381 ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt
     4441 tattcacatt cttgcccgcc tgatgaatgc tcatccggaa ttgcgatcgc atttaaatgg
     4501 ccggccctta attaaggcgc gccgtttaag gatcttcacc tagatccttt taaattaaaa
     4561 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4621 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     4681 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     4741 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     4801 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     4861 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     4921 cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     4981 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5041 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5101 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5161 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5221 ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5281 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5341 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5401 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5461 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5521 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5581 atttccccga aaagtgccac ctgaaacggg cgacggatgg tgatccccct ggccagtgca
     5641 cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa
     5701 agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa
     5761 gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg
     5821 ggaatataaa tgtcaggctc ccttatacac agccagtctg caggtcgatc gaccatagtg
     5881 actggatatg ttgtgtttta cagtattatg tagtctgttt tttatgcaaa atctaattta
     5941 atatattgat atttatatca ttttacgttt ctcgttcaac tttattatac aaagttgtga
     6001 tccacatctt ctgattctat tgatctctca cagtttcaga gaggtttagt tgacaggagt
     6061 tcttgtcatc ctcatgtaac actggataaa gtgtcaagga cacatttgag agtagagcaa
     6121 acaaatagcc acatgcctga aatgaaatcc caacagatta cagatactga atttgatatt
     6181 caaaatcaca aagaaagtgc tgcttctctg ggaaaaggaa gagaattgct tccatgctct
     6241 gctgcagagt caataagcac tgatggaggt ggtctgattt gttctgagga ttcaaattgg
     6301 atggcatgtt acaagaatta cccaactttt taatacaaag ttggcattat aagaaagcat
     6361 tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc
     6421 catccagctg cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa
     6481 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     6541 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga
     6601 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     6661 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     6721 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     6781 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     6841 cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     6901 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     6961 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     7021 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     7081 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     7141 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     7201 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     7261 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     7321 gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg
     7381 gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt
     7441 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     7501 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     7561 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     7621 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     7681 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     7741 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     7801 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     7861 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     7921 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     7981 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     8041 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     8101 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     8161 cctgattctg tggataaccg tattaccgct agccaggaag agtttgtaga aacgcaaaaa
     8221 ggccatccgt caggatggcc ttctgcttag tttgatgcct ggcagtttat ggcgggcgtc
     8281 ctgcccgcca ccctccgggc cgttgcttca caacgttcaa atccgctccc ggcggatttg
     8341 tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtcttccg
     8401 actgagcctt tcgttttatt tgatgcctgg cagttcccta ctctcgc
//
