LOCUS       40924_36478        7733 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-01 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7733)
  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  JOURNAL   Unpublished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 7733)
  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 7733)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7733)
  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            "Unpublished"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..7733
                     /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"
     terminator      2857..3109
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    3318..3442
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3467..3497
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    3769..3809
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(3901..4758)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4759..4863)
                     /label=AmpR promoter
     protein_bind    complement(5160..5283)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(5609..5704)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             5828..6634
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      6780..7368
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(7503..7530)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(7622..7708)
                     /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 acagcgctta gagctcgaat ttccccgatc gttcaaacat ttggcaataa
     2881 agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg
     2941 aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt
     3001 tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc
     3061 gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg ggaattctag
     3121 cttcgacaat tattttggac tctcatgttc tcaagctgaa atccaaaaga aacgtgatgt
     3181 tggttcagat ttggaatcac tgacaatgaa gtttcagtca gccggtttgt caccaaggat
     3241 ccaggcttca tcatgggagc cgagccgttc aagtttcaac ttcgactatt tggatccgtc
     3301 ctagtggatc ccccatcaca actttgtata gaaaagttga acgagaaacg taaaatgata
     3361 taaatatcaa tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa
     3421 cacaacatat ccagtcacta tggcggccgc attaggcacc ccaggcttta cactttatgc
     3481 ttccggctcg tataatgtgt ggattttgag ttaggatccg gcgagatttt caggagctaa
     3541 ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca
     3601 tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt
     3661 tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc
     3721 ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattgc gatcgcattt
     3781 aaatggccgg cccttaatta aggcgcgccg tttaaggatc ttcacctaga tccttttaaa
     3841 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     3901 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     3961 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4021 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4081 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4141 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     4201 tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     4261 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     4321 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     4381 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     4441 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     4501 ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     4561 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     4621 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     4681 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     4741 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     4801 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     4861 gcgcacattt ccccgaaaag tgccacctga aacgggcgac ggatggtgat ccccctggcc
     4921 agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt acccggtggt gcatatcggg
     4981 gatgaaagct ggcgcatgat gaccaccgat atggccagtg tgccggtctc cgttatcggg
     5041 gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat taacctgatg
     5101 ttctggggaa tataaatgtc aggctccctt atacacagcc agtctgcagg tcgatcgacc
     5161 atagtgactg gatatgttgt gttttacagt attatgtagt ctgtttttta tgcaaaatct
     5221 aatttaatat attgatattt atatcatttt acgtttctcg ttcaacttta ttatacaaag
     5281 ttgtgatcca catcttctga ttctattgat ctctcacagt ttcagagagg tttagttgac
     5341 aggagttctt gtcatcctca tgtaacactg gataaagtgt caaggacaca tttgagagta
     5401 gagcaaacaa atagccacat gcctgaaatg aaatcccaac agattacaga tactgaattt
     5461 gatattcaaa atcacaaaga aagtgctgct tctctgggaa aaggaagaga attgcttcca
     5521 tgctctgctg cagagtcaat aagcactgat ggaggtggtc tgatttgttc tgaggattca
     5581 aattggatgg catgttacaa gaattaccca actttttaat acaaagttgg cattataaga
     5641 aagcattgct tatcaatttg ttgcaacgaa caggtcacta tcagtcaaaa taaaatcatt
     5701 atttgccatc cagctgcagc tctggcccgt gtctcaaaat ctctgatgtt acattgcaca
     5761 agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag
     5821 gggtgttatg agccatattc aacgggaaac gtcgaggccg cgattaaatt ccaacatgga
     5881 tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat
     5941 ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag
     6001 cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc
     6061 tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc
     6121 gatccccgga aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
     6181 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
     6241 ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt
     6301 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
     6361 agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
     6421 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
     6481 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
     6541 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
     6601 attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta
     6661 acactggcag agcattacgc tgacttgacg ggacggcgca agctcatgac caaaatccct
     6721 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6781 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6841 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6901 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     6961 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     7021 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     7081 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     7141 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     7201 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     7261 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     7321 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     7381 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     7441 ttatcccctg attctgtgga taaccgtatt accgctagcc aggaagagtt tgtagaaacg
     7501 caaaaaggcc atccgtcagg atggccttct gcttagtttg atgcctggca gtttatggcg
     7561 ggcgtcctgc ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc gctcccggcg
     7621 gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt
     7681 cttccgactg agcctttcgt tttatttgat gcctggcagt tccctactct cgc
//
