LOCUS       40924_36658        8053 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-27 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8053)
  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  JOURNAL   Unpublished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 8053)
  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 8053)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8053)
  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            "Unpublished"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..8053
                     /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"
     promoter        1192..1375
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     CDS             1393..1425
                     /codon_start=1
                     /label=T7 tag (gene 10 leader)
                     /note="leader peptide from bacteriophage T7 gene 10"
                     /translation="MASMTGGQQMG"
     protein_bind    1435..1559
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1596..1626
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1680..2336
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             2681..2983
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3027..3151)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      3177..3429
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    3638..3762
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3787..3817
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4089..4129
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4221..5078)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5079..5183)
                     /label=AmpR promoter
     protein_bind    complement(5480..5603)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(5929..6024)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6148..6954
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7100..7688
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(7823..7850)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(7942..8028)
                     /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 taagcttatc
     1081 atgagcggag aattaaggga gtcacgttat gacccccgcc gatgacgcgg gacaagccgt
     1141 tttacgtttg gaactgacag aaccgcaacg ttgaaggagc cactcagccg cgggtttctg
     1201 gagtttaatg agctaagcac atacgtcaga aaccattatt gcgcgttcaa aagtcgccta
     1261 aggtcactat cagctagcaa atatttcttg tcaaaaatgc tccactgacg ttccataaat
     1321 tcccctcggt atccaattag agtctcatat tcactctcaa tccaaataat ctgcaccgaa
     1381 tctagaatga gcatggctag catgactggt ggacagcaaa tgggtgctgt tatcacaagt
     1441 ttgtacaaaa aagctgaacg agaaacgtaa aatgatataa atatcaatat attaaattag
     1501 attttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca gtcactatgg
     1561 cggcccctcg aggggccgca ttaggcaccc caggctttac actttatgct tccggctcgt
     1621 ataatgtgtg gattttgagt taggatccgg cgagattttc aggagctaag gaagctaaaa
     1681 tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac
     1741 attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata
     1801 ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc
     1861 acattcttgc ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg
     1921 agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa
     1981 cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt
     2041 cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga
     2101 atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg
     2161 ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg
     2221 acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtctgtgat ggcttccatg
     2281 tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaaa
     2341 cgcgtggatc cggcttacta aaagccagat aacagtatgc gtatttgcgc gctgattttt
     2401 gcggtataag aatatatact gatatgtata cccgaagtat gtcaaaaaga ggtgtgctat
     2461 gaagcagcgt attacagtga cagttgacag cgacagctat cagttgctca aggcatatat
     2521 gatgtcaata tctccggtct ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt
     2581 gccgaacgct ggaaagcgga aaatcaggaa gggatggctg aggtcgcccg gtttattgaa
     2641 atgaacggct cttttgctga cgagaacagg gactggtgaa atgcagttta aggtttacac
     2701 ctataaaaga gagagccgtt atcgtctgtt tgtggatgta cagagtgata ttattgacac
     2761 gcccgggcga cggatggtga tccccctggc cagtgcacgt ctgctgtcag ataaagtctc
     2821 ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga
     2881 tatggccagt gtgccggtct ccgttatcgg ggaagaagtg gctgatctca gccaccgcga
     2941 aaatgacatc aaaaacgcca ttaacctgat gttctgggga atataaatgt caggctccct
     3001 tatacacagc cagtctgcag gtcgaccata gtgactggat atgttgtgtt ttacagtatt
     3061 atgtagtctg ttttttatgc aaaatctaat ttaatatatt gatatttata tcattttacg
     3121 tttctcgttc agctttcttg tacaaagtgg tgataaccta gagctcgaat ttccccgatc
     3181 gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga
     3241 ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga
     3301 cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga
     3361 tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt
     3421 tactagatcg ggaattctag cttcgacaat tattttggac tctcatgttc tcaagctgaa
     3481 atccaaaaga aacgtgatgt tggttcagat ttggaatcac tgacaatgaa gtttcagtca
     3541 gccggtttgt caccaaggat ccaggcttca tcatgggagc cgagccgttc aagtttcaac
     3601 ttcgactatt tggatccgtc ctagtggatc ccccatcaca actttgtata gaaaagttga
     3661 acgagaaacg taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca
     3721 gactacataa tactgtaaaa cacaacatat ccagtcacta tggcggccgc attaggcacc
     3781 ccaggcttta cactttatgc ttccggctcg tataatgtgt ggattttgag ttaggatccg
     3841 gcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc
     3901 gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa
     3961 tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa
     4021 aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat
     4081 ccggaattgc gatcgcattt aaatggccgg cccttaatta aggcgcgccg tttaaggatc
     4141 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4201 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4261 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4321 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     4381 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     4441 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     4501 gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg
     4561 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     4621 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     4681 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     4741 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4801 atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc
     4861 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4921 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4981 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5041 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5101 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5161 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga aacgggcgac
     5221 ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt
     5281 acccggtggt gcatatcggg gatgaaagct ggcgcatgat gaccaccgat atggccagtg
     5341 tgccggtctc cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca
     5401 aaaacgccat taacctgatg ttctggggaa tataaatgtc aggctccctt atacacagcc
     5461 agtctgcagg tcgatcgacc atagtgactg gatatgttgt gttttacagt attatgtagt
     5521 ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg
     5581 ttcaacttta ttatacaaag ttgtgatcca catcttctga ttctattgat ctctcacagt
     5641 ttcagagagg tttagttgac aggagttctt gtcatcctca tgtaacactg gataaagtgt
     5701 caaggacaca tttgagagta gagcaaacaa atagccacat gcctgaaatg aaatcccaac
     5761 agattacaga tactgaattt gatattcaaa atcacaaaga aagtgctgct tctctgggaa
     5821 aaggaagaga attgcttcca tgctctgctg cagagtcaat aagcactgat ggaggtggtc
     5881 tgatttgttc tgaggattca aattggatgg catgttacaa gaattaccca actttttaat
     5941 acaaagttgg cattataaga aagcattgct tatcaatttg ttgcaacgaa caggtcacta
     6001 tcagtcaaaa taaaatcatt atttgccatc cagctgcagc tctggcccgt gtctcaaaat
     6061 ctctgatgtt acattgcaca agataaaaat atatcatcat gaacaataaa actgtctgct
     6121 tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac gtcgaggccg
     6181 cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc
     6241 gggcaatcag gtgcgacaat ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt
     6301 ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac
     6361 tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat
     6421 gcatggttac tcaccactgc gatccccgga aaaacagcat tccaggtatt agaagaatat
     6481 cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg
     6541 attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa
     6601 tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg
     6661 cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc
     6721 gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt
     6781 tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg
     6841 aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt
     6901 gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaatca
     6961 gaattggtta attggttgta acactggcag agcattacgc tgacttgacg ggacggcgca
     7021 agctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     7081 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     7141 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     7201 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     7261 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     7321 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     7381 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     7441 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     7501 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     7561 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     7621 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     7681 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     7741 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgctagcc
     7801 aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttagtttg
     7861 atgcctggca gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcacaac
     7921 gttcaaatcc gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca
     7981 gataaaacga aaggcccagt cttccgactg agcctttcgt tttatttgat gcctggcagt
     8041 tccctactct cgc
//
