LOCUS       40924_27583        5931 bp DNA     circular SYN 18-DEC-2018
DEFINITION  MISSA donor vector pLC-ccdB, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5931)
  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 5931)
  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 5931)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5931)
  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..5931
                     /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        64..94
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     CDS             147..803
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     terminator      1003..1089
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      1181..1208
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     primer_bind     1276..1292
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1300..1318
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     protein_bind    1326..1425
                     /label=attL1
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             1576..1878
                     /codon_start=1
                     /label=ccdB
                     /note="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRIVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     rep_origin      2031..2619
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     terminator      complement(2787..2830)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      2962..2989
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     protein_bind    complement(3068..3167)
                     /label=attL2
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        complement(3184..3202)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3214..3230)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(3304..4722)
                     /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(4723..5168)
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     rep_origin      complement(5179..5567)
                     /direction=LEFT
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K;
                     requires the R6K initiator protein pi for replication"
     oriT            5738..5847
                     /label=oriT
                     /note="incP origin of transfer"
ORIGIN
        1 ataacttcgt atagcataca ttatacgaag ttatgtcgaa ttacgcatta ggcaccccca
       61 ggctttacac tttatgcttc cggctcgtat aatgtgtgga ttttgagtta ggatcgtcga
      121 gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg
      181 atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta
      241 cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata
      301 agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg
      361 agttccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt
      421 acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg
      481 atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg
      541 cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga
      601 gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca
      661 caatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc
      721 atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact
      781 gcgatgagtg gcagggcggg gcgtaatttt tttaaggcag ttattggtgc gatcctctag
      841 ctagaaattc gttcaagccg acgccgcttc gccggcgtta actcaagcga ttagatgcac
      901 taagcacata attgctcaca gccaaactat caggacggtc gactctagca tggatctcgg
      961 ggacgtctaa ctactaagcg agagtaggga actgccaggc atcaaataaa acgaaaggct
     1021 cagtcggaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     1081 aggacaaatc cgccgggagc ggatttgaac gttgtgaagc aacggcccgg agggtggcgg
     1141 gcaggacgcc cgccataaac tgccaggcat caaactaagc agaaggccat cctgacggat
     1201 ggcctttttg cgtttctaca aactcttcct gttagttagt tacttaagcg ccagggtttt
     1261 cccagtcacg acgttgtaaa acgacggcca gtgcatacga tttaggtgac actatagaag
     1321 ggccccaaat aatgatttta ttttgactga tagtgacctg ttcgttgcaa caaattgata
     1381 agcaatgctt ttttataatg ccaactttgt acaaaaaagc aggcttttta attaacgact
     1441 ttaggtccaa gcttccctca tgagcttact aaaagccaga taacagtatg cgtatttgcg
     1501 cgctgatttt tgcggtataa gaatatatac tgatatgtat acccgaagta tgtcaaaaag
     1561 aggtgtgctt ctagaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt
     1621 ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat agtgatcccc
     1681 ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat
     1741 atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt
     1801 atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac
     1861 ctgatgttct ggggaatata gtgctgatat cgacccaagt accgccacct aacaattcgt
     1921 tcaagccgag atcggcttcc cggccgcgga gttgttcggt aaattgtcac aacgccgccc
     1981 gccccgttcc actgagcgtc agacccggta gaaaagatca aaggatcttc ttgagatcct
     2041 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     2101 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     2161 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     2221 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     2281 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     2341 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     2401 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     2461 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     2521 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     2581 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     2641 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     2701 gattctgtgg ataaccgtat taccgctagc atggatctcg gggacgtcta actactaagc
     2761 gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcggaa gactgggcct
     2821 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag
     2881 cggatttgaa cgttgtgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa
     2941 ctgccaggca tcaaactaag cagaaggcca tcctgacgga tggccttttt gcgtttctac
     3001 aaactcttcc tgttagttag ttacttaagg ctagcggaat tcgacctaaa gtcggcgcgc
     3061 cagctagacc cagctttctt gtacaaagtt ggcattataa gaaagcattg cttatcaatt
     3121 tgttgcaacg aacaggtcac tatcagtcaa aataaaatca ttatttgcca tccagctgca
     3181 ggccctatag tgagtcgtat taccgtaatc atggtcatag ctgtttcctg tgtgaaattg
     3241 ttatccgctc tcatggcggc cgccaatgcc aataggatat cggcattttc ttttgcgttt
     3301 ttatttgtta actgttaatt gtccttgttc aaggatgctg tctttgacaa cagatgtttt
     3361 cttgcctttg atgttcagca ggaagctagg cgcaaacgtt gattgtttgt ctgcgtagaa
     3421 tcctctgttt gtcatatagc ttgtaatcac gacattgttt cctttcgctt gaggtacagc
     3481 gaagtgtgag taagtaaagg ttacatcgtt aggatcaaga tccattttta acacaaggcc
     3541 agttttgttc agcggcttgt atgggccagt taaagaatta gaaacataac caagcatgta
     3601 aatatcgtta gacgtaatgc cgtcaatcgt catttttgat ccgcgcgagt cagtgaacaa
     3661 gtaccatttg ccgttcattt taaagacgtt cgcgcgttca atttcatctg ttactgtgtt
     3721 agatgcaatc agcggtttca tcactttttt cagtgtgtaa tcatcgttta gctcaatcat
     3781 accgagagcg ccgtttgcta actcagccgt gcgtttttta tcgctttgca gaagtttttg
     3841 actttcttga cggaagaatg atgtgctttt gccatagtat gctttgttaa ataaagattc
     3901 ttcgccttgg tagccatctt cagttccagt gtttgcttca aatactaagt atttgtggcc
     3961 tttatcttct acgtagtgag gatctctcag cgtatggttg tcgcctgagc tgtagttgcc
     4021 ttcatcgatg aactgctgta cattttgata cgtttttccg tcaccgtcaa agattgattt
     4081 ataatcctct acaccgttga tgttcaaaga gctgtctgat gctgatacgt taacttgtgc
     4141 agttgtcagt gtttgtttgc cgtaatgttt accggagaaa tcagtgtaga ataaacggat
     4201 ttttccgtca gatgtaaatg tggctgaacc tgaccattct tgtgtttggt cttttaggat
     4261 agaatcattt gcatcgaatt tgtcgctgtc tttaaagacg cggccagcgt ttttccagct
     4321 gtcaatagaa gtttcgccga ctttttgata gaacatgtaa atcgatgtgt catccgcatt
     4381 tttaggatct ccggctaatg caaagacgat gtggtagccg tgatagtttg cgacagtgcc
     4441 gtcagcgttt tgtaatggcc agctgtccca aacgtccagg ccttttgcag aagagatatt
     4501 tttaattgtg gacgaatcga actcaggaac ttgatatttt tcattttttt gctgttcagg
     4561 gatttgcagc atatcatggc gtgtaatatg ggaaatgccg tatgtttcct tatatggctt
     4621 ttggttcgtt tctttcgcaa acgcttgagt tgcgcctcct gccagcagtg cggtagtaaa
     4681 ggttaatact gttgcttgtt ttgcaaactt tttgatgttc atcgttcatg tctccttttt
     4741 tatgtactgt gttagcggtc tgcttcttcc agccctcctg tttgaagatg gcaagttagt
     4801 tacgcacaat aaaaaaagac ctaaaatatg taaggggtga cgccaaagta tacactttgc
     4861 cctttacaca ttttaggtct tgcctgcttt atcagtaaca aacccgcgcg atttactttt
     4921 cgacctcatt ctattagact ctcgtttgga ttgcaactgg tctattttcc tcttttgttt
     4981 gatagaaaat cataaaagga tttgcagact acgggcctaa agaactaaaa aatctatctg
     5041 tttcttttca ttctctgtat tttttatagt ttctgttgca tgggcataaa gttgcctttt
     5101 taatcacaat tcagaaaata tcataatatc tcatttcact aaataatagt gaacggcagg
     5161 tatatgtggt cgagcccatg tcagccgtta agtgttcctg tgtcactcaa aattgctttg
     5221 agaggctcta agggcttctc agtgcgttac atccctggct tgttgtccac aaccgttaaa
     5281 ccttaaaagc tagaaaagcc ttatatattc ttttttttct tataaaactt aaaaccttag
     5341 aggctattta agttgctgat ttatattaat tttattgttc aaacatgaga gcttagtacg
     5401 tgaaacatga gagcttagta cgttagccat gagagcttag tacgttagcc atgagggttt
     5461 agttcgttaa acatgagagc ttagtacgtt aaacatgaga gcttagtacg tgaaacatga
     5521 gagcttagta cgtactatca acaggttgaa ctgctgatct tcagatcctc tacgccggac
     5581 gcatcgtggc cggatcaatt ccgttttccg ctgcataacc ctgcttcggg gtcattatag
     5641 cgattttttc ggtatatcca tcctttttcg cacgatatac aggattttgc caaagggttc
     5701 gtgtagactt tccttggtgt atccaacggc gtcagccggg caggataggt gaagtaggcc
     5761 cacccgcgag cgggtgttcc ttcttcactg tcccttattc gcacctggcg gtgctcaacg
     5821 ggaatcctgc tctgcgaggc tggccggcta ccgccggcgt aacagatgag ggcaagcgga
     5881 tggctgatga aaccaagcca accaggaagg gcagcccacc tatcatcatg g
//
