LOCUS       40924_36493        7757 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-10 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7757)
  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  JOURNAL   Unpublished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 7757)
  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 7757)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7757)
  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            "Unpublished"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..7757
                     /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..2859
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     terminator      2881..3133
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    3342..3466
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3491..3521
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    3793..3833
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(3925..4782)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4783..4887)
                     /label=AmpR promoter
     protein_bind    complement(5184..5307)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(5633..5728)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             5852..6658
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      6804..7392
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(7527..7554)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(7646..7732)
                     /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 acagcgacta caaggatgac gatgacaagg cttagagctc gaatttcccc
     2881 gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg
     2941 atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc
     3001 atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac
     3061 gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct
     3121 atgttactag atcgggaatt ctagcttcga caattatttt ggactctcat gttctcaagc
     3181 tgaaatccaa aagaaacgtg atgttggttc agatttggaa tcactgacaa tgaagtttca
     3241 gtcagccggt ttgtcaccaa ggatccaggc ttcatcatgg gagccgagcc gttcaagttt
     3301 caacttcgac tatttggatc cgtcctagtg gatcccccat cacaactttg tatagaaaag
     3361 ttgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa
     3421 aacagactac ataatactgt aaaacacaac atatccagtc actatggcgg ccgcattagg
     3481 caccccaggc tttacacttt atgcttccgg ctcgtataat gtgtggattt tgagttagga
     3541 tccggcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac
     3601 caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc
     3661 tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa
     3721 gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc
     3781 tcatccggaa ttgcgatcgc atttaaatgg ccggccctta attaaggcgc gccgtttaag
     3841 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     3901 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     3961 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4021 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     4081 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4141 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4201 gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg tgtcacgctc
     4261 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     4321 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4381 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4441 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4501 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata ccgcgccaca
     4561 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4621 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     4681 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     4741 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     4801 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     4861 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgaaacggg
     4921 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa
     4981 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     5041 agtgtgccgg tctccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     5101 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac
     5161 agccagtctg caggtcgatc gaccatagtg actggatatg ttgtgtttta cagtattatg
     5221 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt
     5281 ctcgttcaac tttattatac aaagttgtga tccacatctt ctgattctat tgatctctca
     5341 cagtttcaga gaggtttagt tgacaggagt tcttgtcatc ctcatgtaac actggataaa
     5401 gtgtcaagga cacatttgag agtagagcaa acaaatagcc acatgcctga aatgaaatcc
     5461 caacagatta cagatactga atttgatatt caaaatcaca aagaaagtgc tgcttctctg
     5521 ggaaaaggaa gagaattgct tccatgctct gctgcagagt caataagcac tgatggaggt
     5581 ggtctgattt gttctgagga ttcaaattgg atggcatgtt acaagaatta cccaactttt
     5641 taatacaaag ttggcattat aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc
     5701 actatcagtc aaaataaaat cattatttgc catccagctg cagctctggc ccgtgtctca
     5761 aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc
     5821 tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag
     5881 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
     5941 tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt
     6001 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
     6061 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
     6121 tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga
     6181 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
     6241 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
     6301 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
     6361 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
     6421 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     6481 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     6541 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     6601 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
     6661 atcagaattg gttaattggt tgtaacactg gcagagcatt acgctgactt gacgggacgg
     6721 cgcaagctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     6781 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     6841 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     6901 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     6961 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     7021 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     7081 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     7141 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     7201 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     7261 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     7321 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     7381 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     7441 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgct
     7501 agccaggaag agtttgtaga aacgcaaaaa ggccatccgt caggatggcc ttctgcttag
     7561 tttgatgcct ggcagtttat ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca
     7621 caacgttcaa atccgctccc ggcggatttg tcctactcag gagagcgttc accgacaaac
     7681 aacagataaa acgaaaggcc cagtcttccg actgagcctt tcgttttatt tgatgcctgg
     7741 cagttcccta ctctcgc
//
