LOCUS       40924_36608        8044 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gateway recycling vector pRED56-MD8-Pnos-12 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8044)
  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  JOURNAL   Unpublished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 8044)
  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 8044)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8044)
  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            "Unpublished"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..8044
                     /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..1416
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     protein_bind    1426..1550
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1587..1617
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             1671..2327
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     CDS             2672..2974
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     protein_bind    complement(3018..3142)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      3168..3420
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    3629..3753
                     /label=attR4
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        3778..3808
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    4080..4120
                     /label=rare cutter sites
                     /note="rare cutter sites"
     CDS             complement(4212..5069)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5070..5174)
                     /label=AmpR promoter
     protein_bind    complement(5471..5594)
                     /label=attR3
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(5920..6015)
                     /label=attL6
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             6139..6945
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      7091..7679
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(7814..7841)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     terminator      complement(7933..8019)
                     /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 gcgactacaa ggatgacgat gacaaggctg ttatcacaag tttgtacaaa
     1441 aaagctgaac gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat
     1501 aaaaaacaga ctacataata ctgtaaaaca caacatatcc agtcactatg gcggcccctc
     1561 gaggggccgc attaggcacc ccaggcttta cactttatgc ttccggctcg tataatgtgt
     1621 ggattttgag ttaggatccg gcgagatttt caggagctaa ggaagctaaa atggagaaaa
     1681 aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg
     1741 catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct
     1801 ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg
     1861 cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga
     1921 tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat
     1981 cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg
     2041 tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt
     2101 tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg
     2161 acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc
     2221 tgatgccgct ggcgattcag gttcatcatg ccgtctgtga tggcttccat gtcggcagaa
     2281 tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa acgcgtggat
     2341 ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa
     2401 gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtgtgcta tgaagcagcg
     2461 tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat
     2521 atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc
     2581 tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc
     2641 tcttttgctg acgagaacag ggactggtga aatgcagttt aaggtttaca cctataaaag
     2701 agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg
     2761 acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact
     2821 ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag
     2881 tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat
     2941 caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc ttatacacag
     3001 ccagtctgca ggtcgaccat agtgactgga tatgttgtgt tttacagtat tatgtagtct
     3061 gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt
     3121 cagctttctt gtacaaagtg gtgataacct agagctcgaa tttccccgat cgttcaaaca
     3181 tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg attatcatat
     3241 aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg acgttattta
     3301 tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg atagaaaaca
     3361 aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg ttactagatc
     3421 gggaattcta gcttcgacaa ttattttgga ctctcatgtt ctcaagctga aatccaaaag
     3481 aaacgtgatg ttggttcaga tttggaatca ctgacaatga agtttcagtc agccggtttg
     3541 tcaccaagga tccaggcttc atcatgggag ccgagccgtt caagtttcaa cttcgactat
     3601 ttggatccgt cctagtggat cccccatcac aactttgtat agaaaagttg aacgagaaac
     3661 gtaaaatgat ataaatatca atatattaaa ttagattttg cataaaaaac agactacata
     3721 atactgtaaa acacaacata tccagtcact atggcggccg cattaggcac cccaggcttt
     3781 acactttatg cttccggctc gtataatgtg tggattttga gttaggatcc ggcgagattt
     3841 tcaggagcta aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata
     3901 tcccaatggc atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat
     3961 aaccagaccg ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac
     4021 aagttttatc cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaattg
     4081 cgatcgcatt taaatggccg gcccttaatt aaggcgcgcc gtttaaggat cttcacctag
     4141 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4201 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4261 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4321 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4381 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4441 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4501 ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg
     4561 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     4621 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     4681 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     4741 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     4801 ccgagttgct cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta
     4861 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     4921 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4981 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5041 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5101 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5161 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg aaacgggcga cggatggtga
     5221 tccccctggc cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg
     5281 tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct
     5341 ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca
     5401 ttaacctgat gttctgggga atataaatgt caggctccct tatacacagc cagtctgcag
     5461 gtcgatcgac catagtgact ggatatgttg tgttttacag tattatgtag tctgtttttt
     5521 atgcaaaatc taatttaata tattgatatt tatatcattt tacgtttctc gttcaacttt
     5581 attatacaaa gttgtgatcc acatcttctg attctattga tctctcacag tttcagagag
     5641 gtttagttga caggagttct tgtcatcctc atgtaacact ggataaagtg tcaaggacac
     5701 atttgagagt agagcaaaca aatagccaca tgcctgaaat gaaatcccaa cagattacag
     5761 atactgaatt tgatattcaa aatcacaaag aaagtgctgc ttctctggga aaaggaagag
     5821 aattgcttcc atgctctgct gcagagtcaa taagcactga tggaggtggt ctgatttgtt
     5881 ctgaggattc aaattggatg gcatgttaca agaattaccc aactttttaa tacaaagttg
     5941 gcattataag aaagcattgc ttatcaattt gttgcaacga acaggtcact atcagtcaaa
     6001 ataaaatcat tatttgccat ccagctgcag ctctggcccg tgtctcaaaa tctctgatgt
     6061 tacattgcac aagataaaaa tatatcatca tgaacaataa aactgtctgc ttacataaac
     6121 agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcgaggcc gcgattaaat
     6181 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
     6241 ggtgcgacaa tctatcgctt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
     6301 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
     6361 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     6421 ctcaccactg cgatccccgg aaaaacagca ttccaggtat tagaagaata tcctgattca
     6481 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
     6541 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
     6601 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     6661 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat
     6721 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
     6781 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
     6841 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
     6901 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc agaattggtt
     6961 aattggttgt aacactggca gagcattacg ctgacttgac gggacggcgc aagctcatga
     7021 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     7081 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     7141 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     7201 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     7261 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     7321 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     7381 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     7441 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     7501 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     7561 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     7621 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     7681 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     7741 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgctagc caggaagagt
     7801 ttgtagaaac gcaaaaaggc catccgtcag gatggccttc tgcttagttt gatgcctggc
     7861 agtttatggc gggcgtcctg cccgccaccc tccgggccgt tgcttcacaa cgttcaaatc
     7921 cgctcccggc ggatttgtcc tactcaggag agcgttcacc gacaaacaac agataaaacg
     7981 aaaggcccag tcttccgact gagcctttcg ttttatttga tgcctggcag ttccctactc
     8041 tcgc
//
