LOCUS       40924_28616        6817 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Expression vector ploxP_cPeUMPS2 DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6817)
  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  JOURNAL   Unpublished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 6817)
  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 6817)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 6817)
  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            "Unpublished"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..6817
                     /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"
     misc_feature    677..846
                     /label=partial sequence of UBL5
                     /note="partial sequence of UBL5"
     misc_feature    847..880
                     /label=loxP
                     /note="loxP"
     protein_bind    847..880
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     regulatory      881..1670
                     /note="includes promoter region of Pseudochoricystis
                     ellipsoidea nuclear gene RBCS for ribulose bisphosphate 
                     carboxylase/oxygenase small subunit"
                     /regulatory_class="promoter"
     misc_feature    1671..1955
                     /note="include intron 1 of Pseudochoricystis ellipsoidea
                     nuclear gene RBCS for ribulose bisphosphate 
                     carboxylase/oxygenase small subunit"
     CDS             1956..3458
                     /codon_start=1
                     /gene="UMPS"
                     /product="uridine monophosphate synthase"
                     /label=UMPS
                     /protein_id="BBB04237.1"
                     /translation="MATSTPSVATAAELKPKEGPITSQEFEELVLRLHEIEAVKFGNFK
                     LKSGLMSPIYIDLRVIVSYPDVLRRVAEVMWHQVSGAAFDVMCGVPYTALPIATCMSLL
                     HGTPMLMRRKEVKEYGTKKAIEGAFKKGQTCLIVEDLVTSGASVMETVEPLEVEGLKVT
                     DVVVLIDREQGGAARMASNGLRLHSAFTLSFIIKTLQAHGLVSAKVADSVAAFIAANQT
                     FTPSAAAPTPSPAAPAQPKRLPFEERASLCQNAAGRKLLELMARKRTNLAVAADVATVE
                     EMLRIADAAGPHIAVFKTHVDIFDKWDDGIATQLRHLADKHEFLIFEDRKFADIGNTVV
                     SQYGGGIYKIADWSDITNAHLVPGPGIIDGLRKVGQEKGRGLLLLAEMSSKGALATGAY
                     TEKVAEAAAANQDFVMGFICQSPAKWATPVPPGLVHMTPGVQLASGSDALGQQYNTPAS
                     VIGQGGSDVIIVGRGIIKAADPAAAAAQYREAGWAAYEATLA"
     gene            1956..3458
                     /gene="UMPS"
                     /label=UMPS
     regulatory      3459..3990
                     /note="includes terminator region of Pseudochoricystis 
                     ellipsoidea nuclear gene RBCS for ribulose bisphosphate 
                     carboxylase/oxygenase small subunit"
                     /regulatory_class="terminator"
     protein_bind    complement(3991..4024)
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4576..4592)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(4629..4647)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4668..4684)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4692..4708)
                     /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(4716..4746)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4761..4782)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5070..5658)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5832..6689)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(6690..6794)
                     /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 gaggtcatga ttgaagtgat tctcaacgat cgtcttggaa agaaagtccg
      721 cgtcaagtgc aagtaagacc cctggtcaca actactggga agtcccacct tcatgtactc
      781 cctttgtact tgtgcaaccg agccatgcac aacacatgtt gtgttagctg ttgctaattg
      841 cactttataa cttcgtatag catacattat acgaagttat atgccctcaa tcggccgatt
      901 tcatgcatga agaagaagat gatgattaca catcattcca gccatctagg gctgccatca
      961 gtaattgcat gacccgcaac ttcccagtca gtcaaatgca caatagaagc atagttgcat
     1021 gggaatcaac ccagcagcct cccgcagagc tagcctaggt acgtgtacgc agccacataa
     1081 tcatcatgaa tgattctgca cggtgctatg atagcaacag atactcgcca gtgcgcacgc
     1141 aagcaatctc atcacaccac tgtggtcagg caagcacgat atagatgtca aatgacagct
     1201 ccattgggag tgcaagtcta ctagagtgca agcctactag tagtaggaag gtccagcgca
     1261 tgccaaaact cctgctaagg ggttgcccaa gccccttggc tggccaaaag tccttaatat
     1321 tttgctaggt aaaggggatc ttcccggagg tggtgttgat gaacatttgt acaccagcat
     1381 ccgggtcctt ttggactctg aaaacgtgag cgagcagcga ctgcttgcag cttccaaacg
     1441 aaaggtctgg aagcgctgga gtggtgagtc aggggtgact cagcaaacac caaggacaca
     1501 cgttttcacc ctaccctcag aacccttgaa ataggggtcg atttccctct aacttaagtg
     1561 gttatccaca aactcacgac gataagagcc agcgccagca ccatttcgtt cctcattttc
     1621 agctgaaagc aaagcctttg catacttatc gctctcagat tacaatcgca atgggtacgc
     1681 agctttctca ctccctcacc gcgtgtggtt cactcaggtt tctggctaag gaaagtctga
     1741 tgtgactttc cttagacgtt aaagcagaat caccccatgc tttaagcagt tttatgtgtc
     1801 ttgcccttca tgtgaagggt ctgtactgtt gcagagtgct ctcttgtgtt cttgagtgtt
     1861 cttgtctcca gtgtgcccat taactttatc catgtcttat gtgtccagga ctgcgccaaa
     1921 gaaactgacg ctgcgctgtt ttgctttgtg cagccatggc aacttcaact ccctcagtgg
     1981 ccacagctgc ggagctgaaa ccaaaggaag ggcctatcac gtcacaggag tttgaagagc
     2041 tcgtgctgcg tttgcatgaa atcgaggctg tcaaattcgg caacttcaag ctcaagagcg
     2101 ggctcatgtc gccgatctac atcgacctgc gcgtcatcgt gtcctatccg gacgtcctgc
     2161 gcagggtggc ggaggtgatg tggcatcagg tgtcgggcgc tgcatttgat gtgatgtgtg
     2221 gcgtgccgta cacggcgctg cccatagcca catgcatgtc gctgctgcac ggcacgccca
     2281 tgctgatgcg gcgcaaggag gtcaaggagt acggcaccaa gaaggccatt gagggcgcct
     2341 tcaagaaggg ccagacctgc ctcattgtgg aggacctggt caccagcggc gcctccgtca
     2401 tggagactgt ggagcccctc gaggtggagg ggctgaaggt gacggatgtg gtggtgctca
     2461 tagaccggga gcagggcggc gcggcgcgca tggccagcaa cgggctgcga ctgcactccg
     2521 cgttcacgct ctccttcatc atcaagacgc tgcaggcgca cggccttgtc agcgctaagg
     2581 tggccgacag cgtcgctgcc ttcatagccg ccaaccagac cttcaccccc tctgcagccg
     2641 cccccacacc ctcacctgcc gctcctgcac agcccaaacg gctgccattt gaggagcgcg
     2701 caagcctatg tcagaacgca gccggcagga agctgttgga gctgatggcg cgcaagcgga
     2761 ccaacctggc ggtggcggcg gacgtggcga cggtggagga gatgctacgc attgcggacg
     2821 cggccgggcc acacatcgcc gtcttcaaga cgcacgtgga catcttcgac aagtgggatg
     2881 atggcatcgc cacccagctg cgccatctgg ctgacaagca tgagttcttg atattcgagg
     2941 acaggaagtt tgcggacatc ggtaacacag tggtgtcgca gtacggcgga ggcatctaca
     3001 agattgccga ctggtcagac atcaccaatg cacacctggt ccctgggcca ggcatcatcg
     3061 atggtctccg taaggtgggc caggagaagg ggcgcggcct gctgttgctg gctgagatga
     3121 gctccaaggg agcccttgcc acaggggcct acaccgagaa ggttgctgaa gcggcagctg
     3181 ccaaccagga ttttgtcatg ggcttcatct gccagtcacc agccaagtgg gccacaccgg
     3241 tccctccagg tctggtgcac atgacaccgg gagtgcagct agcgtctggg agcgacgcat
     3301 tggggcagca gtacaacaca cctgcttctg tcatcggcca gggcggcagc gatgtcatca
     3361 tcgtggggag gggaatcatt aaggcggcag accccgctgc agcggccgca cagtacagag
     3421 aagccggctg ggccgcctat gaggccacgc tggcatgaag cttatcgata ccgtcgacag
     3481 gttctaagca atgacctggg ctgtacatca ttagcaaact gaatctgcag ctttctaggc
     3541 tcttgctgtt cctaggaact tggggttcaa gactgggttt ttgctgtggt tcctgactgc
     3601 agaccatcac tgtctgggtt gtcgtgtcta atgtccatag gtgtgagcac atgctccact
     3661 tcaattccta ctgctgggta actggcgctg tctgagggac ttctgagaag aatgccgaga
     3721 agaacttgag cgatttgtaa ggggatattt tgggtctgta ccaaaggccc agactgggct
     3781 tccagtgcat cgcacatatg catctgcatg tccattcata caatcaacct gttgactgca
     3841 tgacccagat cacaacctcc ttgagttgga agtagcttct tctacagata gaaaatggtg
     3901 catgtcagct gagggttgca ggcgtttaaa agtgcatcaa gtttgcagaa cacatggtga
     3961 ttcatgactg catggaaaca gaccagcaca ataacttcgt atagcataca ttatacgaag
     4021 ttataaacgt ttgattgtct gtggtaaatg ttgctaagtt tcgttgtctc caatcacagc
     4081 gaggacgaca ccatcgggga cctgaagaag ctggtggcag cacagaccgg cacgcgccca
     4141 gagaagattc gcattcagaa aacgtttgat tgtctgtggt aaatgttgct aagtttcgtt
     4201 gtctccaatc acagcgagga cgacaccatc ggggacctga agaagctggt ggcagcacag
     4261 accggcacgc gcccagagaa gattcgcatt cagaagtggt acaatatcta caaggtgcat
     4321 tgctcccgcc tgcttgctgc ctgcaacctg tgaggttgcc agtcctgtcc ctcgtcagat
     4381 caagtcgaaa atgaagtttc tgccactgtc tgcagacaat gcaaaccaag acagggctaa
     4441 aaacctcgac ctacgactgc atgcaggatc acatcacgct ggcagactat gagattcacg
     4501 acggcatggg actggagctg tactacaact aggacggtat cgataagctt gatatcgaat
     4561 tcctgcagcc cgggggatcc actagttcta gagcggccgc caccgcggtg gagctccagc
     4621 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
     4681 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     4741 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4801 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4861 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4921 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4981 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5041 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5101 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5161 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5221 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5281 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5341 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5401 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5461 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     5521 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5581 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5641 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5701 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5761 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5821 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5881 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5941 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6001 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6061 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6121 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6181 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6241 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6301 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6361 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6421 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6481 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6541 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6601 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6661 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6721 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6781 ataggggttc cgcgcacatt tccccgaaaa gtgccac
//
