LOCUS       40924_28611        6127 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector ploxP_cPeUMPS DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6127)
  AUTHORS   Kasai Y, Tsukahara T, Ikeda F, Ide Y, Harayama S.
  TITLE     Improving triacylglycerol production in the unicellular green alga 
            Pseudochoricystis ellipsoidea by metabolic engineering using 
            iterative self-cloning approaches
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6127)
  AUTHORS   Kasai Y, Harayama S.
  TITLE     Direct Submission
  JOURNAL   Submitted (27-OCT-2017) Contact:Yuki Kasai Chuo University, Research
            and Development Initiative; 1-13-27 Kasuga, Bunkyo-ku, Tokyo 
            112-8551, Japan
REFERENCE   3  (bases 1 to 6127)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6127)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (27-OCT-2017) Contact:Yuki Kasai Chuo University, Research and 
            Development Initiative; 1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551, 
            Japan"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6127
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     670..686
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    707..740
                     /label=loxP
                     /note="loxP"
     protein_bind    707..740
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     regulatory      741..1530
                     /note="includes promoter region of Pseudochoricystis
                     ellipsoidea nuclear gene RBCS for ribulose bisphosphate 
                     carboxylase/oxygenase small subunit"
                     /regulatory_class="promoter"
     misc_feature    1531..1815
                     /note="include intron 1 of Pseudochoricystis ellipsoidea
                     nuclear gene RBCS for ribulose bisphosphate 
                     carboxylase/oxygenase small subunit"
     CDS             1816..3318
                     /codon_start=1
                     /gene="UMPS"
                     /product="uridine monophosphate synthase"
                     /label=UMPS
                     /protein_id="BBB04236.1"
                     /translation="MATSTPSVATAAELKPKEGPITSQEFEELVLRLHEIEAVKFGNFK
                     LKSGLMSPIYIDLRVIVSYPDVLRRVAEVMWHQVSGAAFDVMCGVPYTALPIATCMSLL
                     HGTPMLMRRKEVKEYGTKKAIEGAFKKGQTCLIVEDLVTSGASVMETVEPLEVEGLKVT
                     DVVVLIDREQGGAARMASNGLRLHSAFTLSFIIKTLQAHGLVSAKVADSVAAFIAANQT
                     FTPSAAAPTPSPAAPAQPKRLPFEERASLCQNAAGRKLLELMARKRTNLAVAADVATVE
                     EMLRIADAAGPHIAVFKTHVDIFDKWDDGIATQLRHLADKHEFLIFEDRKFADIGNTVV
                     SQYGGGIYKIADWSDITNAHLVPGPGIIDGLRKVGQEKGRGLLLLAEMSSKGALATGAY
                     TEKVAEAAAANQDFVMGFICQSPAKWATPVPPGLVHMTPGVQLASGSDALGQQYNTPAS
                     VIGQGGSDVIIVGRGIIKAADPAAAAAQYREAGWAAYEATLA"
     gene            1816..3318
                     /gene="UMPS"
                     /label=UMPS
     regulatory      3319..3850
                     /note="includes terminator region of Pseudochoricystis 
                     ellipsoidea nuclear gene RBCS for ribulose bisphosphate 
                     carboxylase/oxygenase small subunit"
                     /regulatory_class="terminator"
     protein_bind    complement(3851..3884)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3886..3902)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(3939..3957)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3978..3994)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4002..4018)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(4026..4056)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4071..4092)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4380..4968)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5142..5999)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(6000..6104)
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcataa cttcgtatag
      721 catacattat acgaagttat atgccctcaa tcggccgatt tcatgcatga agaagaagat
      781 gatgattaca catcattcca gccatctagg gctgccatca gtaattgcat gacccgcaac
      841 ttcccagtca gtcaaatgca caatagaagc atagttgcat gggaatcaac ccagcagcct
      901 cccgcagagc tagcctaggt acgtgtacgc agccacataa tcatcatgaa tgattctgca
      961 cggtgctatg atagcaacag atactcgcca gtgcgcacgc aagcaatctc atcacaccac
     1021 tgtggtcagg caagcacgat atagatgtca aatgacagct ccattgggag tgcaagtcta
     1081 ctagagtgca agcctactag tagtaggaag gtccagcgca tgccaaaact cctgctaagg
     1141 ggttgcccaa gccccttggc tggccaaaag tccttaatat tttgctaggt aaaggggatc
     1201 ttcccggagg tggtgttgat gaacatttgt acaccagcat ccgggtcctt ttggactctg
     1261 aaaacgtgag cgagcagcga ctgcttgcag cttccaaacg aaaggtctgg aagcgctgga
     1321 gtggtgagtc aggggtgact cagcaaacac caaggacaca cgttttcacc ctaccctcag
     1381 aacccttgaa ataggggtcg atttccctct aacttaagtg gttatccaca aactcacgac
     1441 gataagagcc agcgccagca ccatttcgtt cctcattttc agctgaaagc aaagcctttg
     1501 catacttatc gctctcagat tacaatcgca atgggtacgc agctttctca ctccctcacc
     1561 gcgtgtggtt cactcaggtt tctggctaag gaaagtctga tgtgactttc cttagacgtt
     1621 aaagcagaat caccccatgc tttaagcagt tttatgtgtc ttgcccttca tgtgaagggt
     1681 ctgtactgtt gcagagtgct ctcttgtgtt cttgagtgtt cttgtctcca gtgtgcccat
     1741 taactttatc catgtcttat gtgtccagga ctgcgccaaa gaaactgacg ctgcgctgtt
     1801 ttgctttgtg cagccatggc aacttcaact ccctcagtgg ccacagctgc ggagctgaaa
     1861 ccaaaggaag ggcctatcac gtcacaggag tttgaagagc tcgtgctgcg tttgcatgaa
     1921 atcgaggctg tcaaattcgg caacttcaag ctcaagagcg ggctcatgtc gccgatctac
     1981 atcgacctgc gcgtcatcgt gtcctatccg gacgtcctgc gcagggtggc ggaggtgatg
     2041 tggcatcagg tgtcgggcgc tgcatttgat gtgatgtgtg gcgtgccgta cacggcgctg
     2101 cccatagcca catgcatgtc gctgctgcac ggcacgccca tgctgatgcg gcgcaaggag
     2161 gtcaaggagt acggcaccaa gaaggccatt gagggcgcct tcaagaaggg ccagacctgc
     2221 ctcattgtgg aggacctggt caccagcggc gcctccgtca tggagactgt ggagcccctc
     2281 gaggtggagg ggctgaaggt gacggatgtg gtggtgctca tagaccggga gcagggcggc
     2341 gcggcgcgca tggccagcaa cgggctgcga ctgcactccg cgttcacgct ctccttcatc
     2401 atcaagacgc tgcaggcgca cggccttgtc agcgctaagg tggccgacag cgtcgctgcc
     2461 ttcatagccg ccaaccagac cttcaccccc tctgcagccg cccccacacc ctcacctgcc
     2521 gctcctgcac agcccaaacg gctgccattt gaggagcgcg caagcctatg tcagaacgca
     2581 gccggcagga agctgttgga gctgatggcg cgcaagcgga ccaacctggc ggtggcggcg
     2641 gacgtggcga cggtggagga gatgctacgc attgcggacg cggccgggcc acacatcgcc
     2701 gtcttcaaga cgcacgtgga catcttcgac aagtgggatg atggcatcgc cacccagctg
     2761 cgccatctgg ctgacaagca tgagttcttg atattcgagg acaggaagtt tgcggacatc
     2821 ggtaacacag tggtgtcgca gtacggcgga ggcatctaca agattgccga ctggtcagac
     2881 atcaccaatg cacacctggt ccctgggcca ggcatcatcg atggtctccg taaggtgggc
     2941 caggagaagg ggcgcggcct gctgttgctg gctgagatga gctccaaggg agcccttgcc
     3001 acaggggcct acaccgagaa ggttgctgaa gcggcagctg ccaaccagga ttttgtcatg
     3061 ggcttcatct gccagtcacc agccaagtgg gccacaccgg tccctccagg tctggtgcac
     3121 atgacaccgg gagtgcagct agcgtctggg agcgacgcat tggggcagca gtacaacaca
     3181 cctgcttctg tcatcggcca gggcggcagc gatgtcatca tcgtggggag gggaatcatt
     3241 aaggcggcag accccgctgc agcggccgca cagtacagag aagccggctg ggccgcctat
     3301 gaggccacgc tggcatgaag cttatcgata ccgtcgacag gttctaagca atgacctggg
     3361 ctgtacatca ttagcaaact gaatctgcag ctttctaggc tcttgctgtt cctaggaact
     3421 tggggttcaa gactgggttt ttgctgtggt tcctgactgc agaccatcac tgtctgggtt
     3481 gtcgtgtcta atgtccatag gtgtgagcac atgctccact tcaattccta ctgctgggta
     3541 actggcgctg tctgagggac ttctgagaag aatgccgaga agaacttgag cgatttgtaa
     3601 ggggatattt tgggtctgta ccaaaggccc agactgggct tccagtgcat cgcacatatg
     3661 catctgcatg tccattcata caatcaacct gttgactgca tgacccagat cacaacctcc
     3721 ttgagttgga agtagcttct tctacagata gaaaatggtg catgtcagct gagggttgca
     3781 ggcgtttaaa agtgcatcaa gtttgcagaa cacatggtga ttcatgactg catggaaaca
     3841 gaccagcaca ataacttcgt atagcataca ttatacgaag ttatggatcc actagttcta
     3901 gagcggccgc caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaattgcg
     3961 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4021 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4081 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4141 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4201 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4261 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4321 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4381 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4441 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4501 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4561 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4621 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4681 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4741 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4801 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4861 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4921 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4981 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5041 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5101 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5161 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5221 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5281 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5341 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5401 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5461 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5521 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5581 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5641 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5701 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5761 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5821 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5881 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5941 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6001 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6061 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6121 gtgccac
//
