LOCUS       40924_36928        5917 bp DNA     circular SYN 18-DEC-2018
DEFINITION  MISSA donor vector pRG-ccdB, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5917)
  AUTHORS   Chen QJ, Xie M, Ma XX, Dong L, Chen J, Wang XC.
  TITLE     MISSA is a highly efficient in vivo DNA assembly method for plant 
            multiple-gene transformation
  JOURNAL   Plant Physiol. 153 (1), 41-51 (2010)
  PUBMED    20200068
REFERENCE   2  (bases 1 to 5917)
  AUTHORS   Chen Q-J., Xie M, Ma X-X., Dong L, Chen J, Wang X-C.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-JAN-2010) State Key Laboratory of Plant Physiology and
            Biochemistry, College of Biological Sciences, China Agricultural 
            University, No. 2 West Yuanmingyuan Road, Haidian, Beijing 100193, 
            China
REFERENCE   3  (bases 1 to 5917)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5917)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant 
            Physiol."; date: "2010"; volume: "153"; issue: "1"; pages: "41-51"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (27-JAN-2010) State Key Laboratory of Plant Physiology and 
            Biochemistry, College of Biological Sciences, China Agricultural 
            University, No. 2 West Yuanmingyuan Road, Haidian, Beijing 100193, 
            China"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5917
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(1..34)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        73..101
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             290..820
                     /codon_start=1
                     /label=GmR
                     /note="gentamycin acetyltransferase"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPRFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     terminator      919..1005
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      1097..1124
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     primer_bind     1192..1208
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1216..1234
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     protein_bind    complement(1249..1373)
                     /label=attR2
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1533..1835
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRIVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     rep_origin      1988..2576
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(2744..2787)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      2919..2946
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     protein_bind    3029..3153
                     /label=attR1
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(3170..3188)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3200..3216)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(3290..4708)
                     /codon_start=1
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        complement(4709..5154)
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     rep_origin      complement(5165..5553)
                     /direction=LEFT
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     oriT            5724..5833
                     /label=oriT
                     /note="incP origin of transfer"
ORIGIN
        1 ataacttcgt atagcataca ttatacgaag ttatgtcgaa caccgtggaa acggatgaag
       61 gcacgaaccc agttgacata agcctgttcg gttcgtaaac tgtaatgcaa gtagcgtatg
      121 cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg
      181 gttttcatgg cttgttatga ctgttttttt gtacagtcta tgcctcgggc atccaagcag
      241 caagcgcgtt acgccgtggg tcgatgtttg atgttatgga gcagcaacga tgttacgcag
      301 cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaggtg gctcaagtat
      361 gggcatcatt cgcacatgta ggctcggccc tgaccaagtc aaatccatgc gggctgctct
      421 tgatcttttc ggtcgtgagt tcggagacgt agccacctac tcccaacatc agccggactc
      481 cgattacctc gggaacttgc tccgtagtaa gacattcatc gcgcttgctg ccttcgacca
      541 agaagcggtt gttggcgctc tcgcggctta cgttctgccc aggtttgagc agccgcgtag
      601 tgagatctat atctatgatc tcgcagtctc cggcgagcac cggaggcagg gcattgccac
      661 cgcgctcatc aatctcctca agcatgaggc caacgcgctt ggtgcttatg tgatctacgt
      721 gcaagcagat tacggtgacg atcccgcagt ggctctctat acaaagttgg gcatacggga
      781 agaagtgatg cactttgata tcgacccaag taccgccacc taacaattcg ttcaagccga
      841 gatcggcttc ccggtcgact ctagcatgga tctcggggac gtctaactac taagcgagag
      901 tagggaactg ccaggcatca aataaaacga aaggctcagt cggaagactg ggcctttcgt
      961 tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat
     1021 ttgaacgttg tgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc
     1081 aggcatcaaa ctaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact
     1141 cttcctgtta gttagttact taagcgccag ggttttccca gtcacgacgt tgtaaaacga
     1201 cggccagtgc atacgattta ggtgacacta tagaagggcc caatcgacca tagtgactgg
     1261 atatgttgtg ttttacagta ttatgtagtc tgttttttat gcaaaatcta atttaatata
     1321 ttgatattta tatcatttta cgtttctcgt tcagctttct tgtacaaagt ggttcgataa
     1381 ttccttaatt aacgacttta ggtccaagct tccctcatga gcttactaaa agccagataa
     1441 cagtatgcgt atttgcgcgc tgatttttgc ggtataagaa tatatactga tatgtatacc
     1501 cgaagtatgt caaaaagagg tgtgcttcta gaatgcagtt taaggtttac acctataaaa
     1561 gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac acgcccgggc
     1621 gacggatagt gatccccctg gccagtgcac gtctgctgtc agataaagtc tcccgtgaac
     1681 tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca
     1741 gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca
     1801 tcaaaaacgc cattaacctg atgttctggg gaatatagtg ctgatatcga cccaagtacc
     1861 gccacctaac aattcgttca agccgagatc ggcttcccgg ccgcggagtt gttcggtaaa
     1921 ttgtcacaac gccgcccgcc ccgttccact gagcgtcaga cccggtagaa aagatcaaag
     1981 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2041 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2101 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     2161 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2221 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     2281 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     2341 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     2401 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     2461 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2521 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2581 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     2641 ttcctgcgtt atcccctgat tctgtggata accgtattac cgctagcatg gatctcgggg
     2701 acgtctaact actaagcgag agtagggaac tgccaggcat caaataaaac gaaaggctca
     2761 gtcggaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag
     2821 gacaaatccg ccgggagcgg atttgaacgt tgtgaagcaa cggcccggag ggtggcgggc
     2881 aggacgcccg ccataaactg ccaggcatca aactaagcag aaggccatcc tgacggatgg
     2941 cctttttgcg tttctacaaa ctcttcctgt tagttagtta cttaaggcta gcggaattcg
     3001 acctaaagtc ggcgcgccaa gctatcaaac aagtttgtac aaaaaagctg aacgagaaac
     3061 gtaaaatgat ataaatatca atatattaaa ttagattttg cataaaaaac agactacata
     3121 atactgtaaa acacaacata tccagtcact atggcggccg gctgcaggcc ctatagtgag
     3181 tcgtattacc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctctcat
     3241 ggcggccgcc aatgccaata ggatatcggc attttctttt gcgtttttat ttgttaactg
     3301 ttaattgtcc ttgttcaagg atgctgtctt tgacaacaga tgttttcttg cctttgatgt
     3361 tcagcaggaa gctaggcgca aacgttgatt gtttgtctgc gtagaatcct ctgtttgtca
     3421 tatagcttgt aatcacgaca ttgtttcctt tcgcttgagg tacagcgaag tgtgagtaag
     3481 taaaggttac atcgttagga tcaagatcca tttttaacac aaggccagtt ttgttcagcg
     3541 gcttgtatgg gccagttaaa gaattagaaa cataaccaag catgtaaata tcgttagacg
     3601 taatgccgtc aatcgtcatt tttgatccgc gcgagtcagt gaacaagtac catttgccgt
     3661 tcattttaaa gacgttcgcg cgttcaattt catctgttac tgtgttagat gcaatcagcg
     3721 gtttcatcac ttttttcagt gtgtaatcat cgtttagctc aatcataccg agagcgccgt
     3781 ttgctaactc agccgtgcgt tttttatcgc tttgcagaag tttttgactt tcttgacgga
     3841 agaatgatgt gcttttgcca tagtatgctt tgttaaataa agattcttcg ccttggtagc
     3901 catcttcagt tccagtgttt gcttcaaata ctaagtattt gtggccttta tcttctacgt
     3961 agtgaggatc tctcagcgta tggttgtcgc ctgagctgta gttgccttca tcgatgaact
     4021 gctgtacatt ttgatacgtt tttccgtcac cgtcaaagat tgatttataa tcctctacac
     4081 cgttgatgtt caaagagctg tctgatgctg atacgttaac ttgtgcagtt gtcagtgttt
     4141 gtttgccgta atgtttaccg gagaaatcag tgtagaataa acggattttt ccgtcagatg
     4201 taaatgtggc tgaacctgac cattcttgtg tttggtcttt taggatagaa tcatttgcat
     4261 cgaatttgtc gctgtcttta aagacgcggc cagcgttttt ccagctgtca atagaagttt
     4321 cgccgacttt ttgatagaac atgtaaatcg atgtgtcatc cgcattttta ggatctccgg
     4381 ctaatgcaaa gacgatgtgg tagccgtgat agtttgcgac agtgccgtca gcgttttgta
     4441 atggccagct gtcccaaacg tccaggcctt ttgcagaaga gatattttta attgtggacg
     4501 aatcgaactc aggaacttga tatttttcat ttttttgctg ttcagggatt tgcagcatat
     4561 catggcgtgt aatatgggaa atgccgtatg tttccttata tggcttttgg ttcgtttctt
     4621 tcgcaaacgc ttgagttgcg cctcctgcca gcagtgcggt agtaaaggtt aatactgttg
     4681 cttgttttgc aaactttttg atgttcatcg ttcatgtctc cttttttatg tactgtgtta
     4741 gcggtctgct tcttccagcc ctcctgtttg aagatggcaa gttagttacg cacaataaaa
     4801 aaagacctaa aatatgtaag gggtgacgcc aaagtataca ctttgccctt tacacatttt
     4861 aggtcttgcc tgctttatca gtaacaaacc cgcgcgattt acttttcgac ctcattctat
     4921 tagactctcg tttggattgc aactggtcta ttttcctctt ttgtttgata gaaaatcata
     4981 aaaggatttg cagactacgg gcctaaagaa ctaaaaaatc tatctgtttc ttttcattct
     5041 ctgtattttt tatagtttct gttgcatggg cataaagttg cctttttaat cacaattcag
     5101 aaaatatcat aatatctcat ttcactaaat aatagtgaac ggcaggtata tgtggtcgag
     5161 cccatgtcag ccgttaagtg ttcctgtgtc actcaaaatt gctttgagag gctctaaggg
     5221 cttctcagtg cgttacatcc ctggcttgtt gtccacaacc gttaaacctt aaaagctaga
     5281 aaagccttat atattctttt ttttcttata aaacttaaaa ccttagaggc tatttaagtt
     5341 gctgatttat attaatttta ttgttcaaac atgagagctt agtacgtgaa acatgagagc
     5401 ttagtacgtt agccatgaga gcttagtacg ttagccatga gggtttagtt cgttaaacat
     5461 gagagcttag tacgttaaac atgagagctt agtacgtgaa acatgagagc ttagtacgta
     5521 ctatcaacag gttgaactgc tgatcttcag atcctctacg ccggacgcat cgtggccgga
     5581 tcaattccgt tttccgctgc ataaccctgc ttcggggtca ttatagcgat tttttcggta
     5641 tatccatcct ttttcgcacg atatacagga ttttgccaaa gggttcgtgt agactttcct
     5701 tggtgtatcc aacggcgtca gccgggcagg ataggtgaag taggcccacc cgcgagcggg
     5761 tgttccttct tcactgtccc ttattcgcac ctggcggtgc tcaacgggaa tcctgctctg
     5821 cgaggctggc cggctaccgc cggcgtaaca gatgagggca agcggatggc tgatgaaacc
     5881 aagccaacca ggaagggcag cccacctatc atcatgg
//
