LOCUS       sequence-296746-        9315 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Over-expression of heterologus squalene epoxidase from 
            Nannochloropsis oceanica.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9315)
  AUTHORS   Jaramillo-Madrid AC, Abbriano R, Ashworth J, Fabris M, Pernice M, 
            Ralph PJ
  TITLE     Overexpression of Key Sterol Pathway Enzymes in Two Model Marine 
            Diatoms Alters Sterol Profiles in Phaeodactylum tricornutum.
  JOURNAL   Pharmaceuticals (Basel). 2020 Dec 21;13(12). pii: ph13120481. doi: 
            10.3390/ph13120481.
  PUBMED    33371196
REFERENCE   2  (bases 1 to 9315)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Pharmaceuticals (Basel)."; date: "2020-12-21"; volume: "13(12). 
            pii: ph13120481. doi"; pages: " 10.3390/ph13120481"
COMMENT     Sequence Label: pL2-1 ConjEpi TpEF2nat TpEF2NoSQEVenusThrFLAG
FEATURES             Location/Qualifiers
     source          1..9315
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1181..1210
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1184..1213
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1187..1216
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             join(2620..2622,2623..2625,2626..3138)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIE"
     primer_bind     complement(2665..2686)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2926..2945)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3273..3294
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3334..3351
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3352..3369
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3370..3393
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     rep_origin      complement(3908..4496)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3988..4007)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4587..5378)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     misc_feature    5857..6360
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        6361..6547
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             6548..7210
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     CDS             complement(7368..7739)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELVRINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            7772..7881
                     /note="incP origin of transfer"
     CDS             8471..9037
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFT
                     LREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRR
                     LTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTL
                     CGLDTALYDGTASDGEQALYMSMPCP"
ORIGIN
        1 ttgacaaaca catagtagtg tcccgataat cccaattgaa aaacatagta agcgatagac
       61 tctctttcac tctgcttttc tctgctctct ttttttgacg gttgacagac atacttccac
      121 gtggccgacg taactcgttg agcggtttgt cactatatgt tcatgtctcg aaaagatgac
      181 gggacaccaa gattcctgct tgcttctacc cgttgacgcc gcacgcacca acaaagacat
      241 caacgcccgc ctccacccag ccactttaca aagtccttct tttcaacacg ctagttagca
      301 cccccgcgat taattaaaga gaaaagaacg tcaagaatgg aggcatcaac accaacacaa
      361 ggacaagtgc cacatagacc atcattgtca tcagagccaa caccaccagg atcaccaaac
      421 tcatcagtgg gagtgggagg atcaattgtg gtggcatcat cattgttgtt ggtgtcatca
      481 ttgacattgt ggcaagcatg gcatcaacat caacaaacac cattggcaac aacaggaaac
      541 gcagtgaaca cagcaaagtt gttgttgcca gtgttgggag tgttggcatc attgtcattt
      601 ttgtgttaca catcattgtt ttcatcagca caatcacaat catcatgtga tgagccagat
      661 acaaagatta cattgggaca aaagtcaaca gattcatcat tttcaccatc aacaacaaca
      721 acatcatcac aattgccatc atcatcaatt tcaaacgcag agatgttgtt tagatcagga
      781 ggagtgggag gaaacagaat tccaccaaga caagcaccat cagcaggaca aacaattatt
      841 gtggtgggag caggaacagc aggagcagca gcagcagtgg gattggcaga gagaggattt
      901 aaggtgttgt tgtttgagag agatatgaca ttgcaagata gaattgtggg agagttgttg
      961 caaccaggag gagtgagagc attggagaga ttgggattgg atgagtgtgc aaaggaggga
     1021 attgattcag tgggagtgga gggatacgtg gtgtttagac cacataagtt ggaggtggag
     1081 ggaggagagg aggaggtggt gaagaacatt atgttgaact acccagcata cgatccagca
     1141 tcaatgaagg agttgtttgg agtgattgat ccatgtgtgt caccatcatc atcatcatca
     1201 tcatcatcat catcattgtt ttcaattcca ccatcaacag aggaggtgca tggagcagga
     1261 aagacattgt caccacattc agcaacaaag gcaagaaaga cagcagcagc agtggcaaca
     1321 gcagcaacaa cagtggcaca agagcatgag ccattgcgtc caaagggaag atcatttcat
     1381 aacggaagat ttgtgcaaag attgagagag agagcattgg cagagaagaa cgtgacagtg
     1441 attcaaggaa ttgtgtcaag attggtgcat gaggaggatt tgacagaggg agcaaaggag
     1501 ggaagagaga agggaaagaa gcaaagagtg gtgggagtgg agtacaagat tgagacagga
     1561 agaacattga caaacgagga gggagtggag gtgccagaga aggagacaag acgtgcattg
     1621 gcaccattga caattgtggc agatggattg tggtcatcat tgagaacaca tgtggataag
     1681 tgtgcaaagc cacatcaatt gtcatcattt gtgggattga ttattacaca tccatttatg
     1741 tcatcaccat tgccacatgc acattgtgga catgtggtgt tggcagagcc atcaccagtg
     1801 ttgttgtacc aaatttcatc aacagagaca agagtgttgg tggatattca tggaaagttg
     1861 ccatcagcat caacaggaga gttggcatca catttgtaca cacatatttt gccacaattg
     1921 ccaccatgtt tgcaagcacc atttgcagag gcagtggcaa gaggaaactt taagtcaatg
     1981 ccaaacagag caatgccagc agcaaagttg gcaaaggatg gagtgatttt gttgggagat
     2041 gcattgaaca tgagacatcc attgacagga ggaggaatga cagtggcatt gagagatgtg
     2101 gaggcattgt gttacttgtt ggaggagcca acagcagatt tgacaaaccc agtggcattt
     2161 agagcattga agaagagatt tgcaacagag agaaagtcat gggcagcaac aattaacgtg
     2221 ttggcaaacg cattgcatgc agtgttttca acaccagatg gagatttgac aagaaaggat
     2281 ttgagagatg catgttttga ttacttgtca attggaggat actgtgcagg aggaccaatt
     2341 aacttgttgt caggattgac accaagacca tcaattttgg caatgcattt ttttttggtg
     2401 gcatttcatg gagtgggaaa gtacgtgtgg ccatttccaa cattgggagg aatggtgaga
     2461 atgtacgata tgttgcatgt ggcatgtcaa attattatgc cattgttgac agtggaggga
     2521 tcaacagtgt tggcatcatg gccagtgaga acagcaattg gagtggtgtt tccatacgga
     2581 aagtcaggta acggagatct gggtggctcc ggcggccgga tggtgagcaa gggcgaggag
     2641 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     2701 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagctg
     2761 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgggctac
     2821 ggcctgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2881 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2941 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     3001 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     3061 agccacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag
     3121 atccgccaca acatcgagga cggcggcgtg cagctcgccg accactacca gcagaacacc
     3181 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccgcc
     3241 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     3301 gccgggatca ctctcggcat ggacgagctg gcgctggtcc ctcgcggtag ccaccaccac
     3361 catcaccacg actacaagga tgatgacgat aagtgagctt atatcttctt gcaacaatgg
     3421 tagcgcgatt gatgagataa tagataaaaa aatccactgt gaatgttttt caatgagtct
     3481 acaatagtga cttgacaggt cgtatagagt gagacattag ttatctctaa ccaaccctgc
     3541 tcgctacaga ggaggggaac aaaggattgt gcagtgccat acgacaagtt cgcctcaatc
     3601 gccaaacccc ttgactgtgc ttcccgagcg gaaacattta gattggctac tacaaccgtt
     3661 gctctccggg cggataagag aataaatctg gattcggcag agtagtcggc cttatcatca
     3721 gttagatgtt gataatcccg ttgggcaggt cacacgctag gtaaatgtga gaccacccca
     3781 ggatacatag attaccacaa ctccgagccc ttccacccac agaatcaggg gataacgcag
     3841 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3901 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3961 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4021 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4081 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4141 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4201 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4261 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4321 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     4381 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4441 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4501 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4561 ttttggtcat gcattctagg tgattattat ttgccgacta ccttggtgat ctcgcctttc
     4621 acgtagtgga caaattcttc caactgatct gcgcgcgagg ccaagcgatc ttcttcttgt
     4681 ccaagataag cctgtctagc ttcaagtatg acgggctgat actgggccgg caggcgctcc
     4741 attgcccagt cggcagcgac atccttcggc gcgattttgc cggttactgc gctgtaccaa
     4801 atgcgggaca acgtaagcac tacatttcgc tcatcgccag cccagtcggg cggcgagttc
     4861 catagcgtta aggtttcatt tagcgcctca aatagatcct gttcaggaac cggatcaaag
     4921 agttcctccg ccgctggacc taccaaggca acgctatgtt ctcttgcttt tgtcagcaag
     4981 atagccagat caatgtcgat cgtggctggc tcgaagatac cagcaagaat gtcattgcgc
     5041 tgccattctc caaattgcag ttcgcgctta gctggataac gccacggaat gatgtcgtcg
     5101 tgcacaacaa tggtgacttc tacagcgcgg agaatctcac tctctccagg ggaagccgaa
     5161 gtttccaaaa ggtcgttgat caaagctcgc cgcgttgttt catcaagcct tacggtcacc
     5221 gtaaccagca aatcaatatc actgtgtggc ttcaggccgc catccactgc ggagccgtac
     5281 aaatgtacgg ccagcaacgt cggttcgaga tggcgctcga tgacgccaac tacctctgat
     5341 agttgagtcg atacttcggc gatcaccgct tccctcatgc gaaacgatcc tcatcctgtc
     5401 tcttgatcag atattgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag
     5461 tttactttgc agggcttccc aaccttacca gagggcgccc cagctggcaa ttccggttcg
     5521 cttgctaagc ttgcatgcct gcaagtcgac tctagaggag cggtgatcac aggcagcaac
     5581 gctctgtcat cgttacaatc aacatgctac cctccgcgag atcatccgtg tttcaaaccc
     5641 ggcagcttag ttgccgttct tccgaatagc atcggtaaca tgagcaaagt ctgccgcctt
     5701 acaacggctc tcccgctgac gccgtcccgg actgatgggc tgcctgtatc gagtggtgat
     5761 tttgtgccga gctcattact cgcatccatt ctcaggctgt ctcgtctcgt ctccgaggtc
     5821 tcatactaat gaggagctag ctcgtcttca gcgagcatca cgtgctataa aaataattat
     5881 aatttaaatt ttttaatata aatatataaa ttaaaaatag aaagtaaaaa aagaaattaa
     5941 agaaaaaata gtttttgttt tccgaagatg taaaagactc tagggggatc gccaacaaat
     6001 actacctttt atcttgctcg tcctgctctc aggtattaat gccgaattgt ttcatcttgt
     6061 ctgtgtagaa gaccacacac gaaaatcctg tgattttaca ttttacttat cgttaatcga
     6121 atgtatatct atttaatctg cttttcttgt ctaataaata tatatgtaaa gtacgctttt
     6181 tgttgaaatt ttttaaacct ttgtttattt ttttttcttc attccgtaac tcttctacct
     6241 tctttattta ctttctaaaa tccaaataca aaacataaaa ataaataaac acagagtaaa
     6301 ttcccaaatt attccatcat taaaagatac gaggcgcgtg taagttacag gcaagcgatc
     6361 ctagtacact ctatattttt ttatgcctcg gtaatgattt tcattttttt tttccaccta
     6421 gcggatgact cttttttttt cttagcgatt ggcattatca cataatgaat tatacattat
     6481 ataaagtaat gtgatttctt cgaagaatat actaaaaaat gagcaggcaa gataaacgaa
     6541 ggcaaagatg acagagcaga aagccctagt aaagcgtatt acaaatgaaa ccaagattca
     6601 gattgcgatc tctttaaagg gtggtcccct agcgatagag cactcgatct tcccagaaaa
     6661 agaggcagaa gcagtagcag aacaggccac acaatcgcaa gtgattaacg tccacacagg
     6721 tatagggttt ctggaccata tgatacatgc tctggccaag cattccggct ggtcgctaat
     6781 cgttgagtgc attggtgact tacacataga cgaccatcac accactgaag actgcgggat
     6841 tgctctcggt caagctttta aagaggccct aggggccgtg cgtggagtaa aaaggtttgg
     6901 atcaggattt gcgcctttgg atgaggcact ttccagagcg gtggtagatc tttcgaacag
     6961 gccgtacgca gttgtcgaac ttggtttgca aagggagaaa gtaggagatc tctcttgcga
     7021 gatgatcccg cattttcttg aaagctttgc agaggctagc agaattaccc tccacgttga
     7081 ttgtctgcga ggcaagaatg atcatcaccg tagtgagagt gcgttcaagg ctcttgcggt
     7141 tgccataaga gaagccacct cgcccaatgg taccaacgat gttccctcca ccaaaggtgt
     7201 tcttatgtag ttttacacag gagtctggac ttgaccaatg gatcgtcttg ccttgctcgt
     7261 cggtgatgta cttcaccagc tccgcgaagt cgcccttctt gatggagcgc atggggacgt
     7321 gcttggcaat cacgcgcacc ccccggccgt tttagcggct aaaaaagtca tggctctgcc
     7381 ctcgggcgga ccacgcccat catgaccttg ccaagctcgt cctgcttctc ttcgatcttc
     7441 gccagcaggg cgaggatcgt ggcatcaccg aaccgcgccg tgcgcgggtc gtcggtgagc
     7501 cagagtttca gcaggccgcc caggcggccc aggtcgccat tgatgcggac cagctcgcgg
     7561 acgtgctcat agtccacgac gcccgtgatt ttgtagccct ggccgacggc cagcaggtag
     7621 gccgacaggc tcatgccggc cgccgccgcc ttttcctcaa tcgcccttcg ttcgtctgga
     7681 aggcagtaca ccttgatagg tgggctgccc ttcctggttg gcttggtttc atcagccatc
     7741 cgcttgccct catctgttac gccggcggta gccggccagc ctcgcagagc aggattcccg
     7801 ttgagcaccg ccaggtgcga ataagggaca gtgaagaagg aacacccgct cgcgggtggg
     7861 cctacttcac ctatcctgcc cggctgacgc cgttggatac accaaggaaa gtctacacga
     7921 accctttggc aaaatcctgt atatcgtgcg aaaaaggatg gatataccga aaaaatcgct
     7981 ataatgaccc cgaagcaggg ttatgcagcg gaagatcgct agcaaggaga gtgtgcaatg
     8041 cagtcaattc aatagatatg taaaatgttg atgtaaggga aacaatcaag aacagatttc
     8101 agatcaaact gaatgaactc ttgcactttg acacttgaca aacacatagt agtgtcccga
     8161 taatcccaat tgaaaaacat agtaagcgat agactctctt tcactctgct tttctctgct
     8221 ctcttttttt gacggttgac agacatactt ccacgtggcc gacgtaactc gttgagcggt
     8281 ttgtcactat atgttcatgt ctcgaaaaga tgacgggaca ccaagattcc tgcttgcttc
     8341 tacccgttga cgccgcacgc accaacaaag acatcaacgc ccgcctccac ccagccactt
     8401 tacaaagtcc ttcttttcaa cacgctagtt agcacccccg cgattaatta aagagaaaag
     8461 aacgtcaaga atgaccactc ttgacgacac ggcttaccgg taccgcacca gtgtcccggg
     8521 ggacgccgag gccatcgagg cactggatgg gtccttcacc accgacaccg tattccgcgt
     8581 caccgccacc ggggacggct tcaccctgcg ggaggtgccg gtggacccgc ccctgaccaa
     8641 ggtgttcccc gacgacgaat cggacgacga atcggacgac ggggaggacg gcgacccgga
     8701 ctcccggacg ttcgtcgcgt acggggacga cggcgacctg gcgggcttcg tggtcgtctc
     8761 gtactccggc tggaaccgcc ggctgaccgt cgaggacatc gaggtcgccc cggagcaccg
     8821 ggggcacggg gtcgggcgcg cgttgatggg gctcgcgacg gagttcgccc gcgagcgggg
     8881 cgccgggcac ctctggctgg aggtcaccaa cgtcaacgca ccggcgatcc acgcgtaccg
     8941 gcggatgggg ttcaccctct gcggcctgga caccgccctg tacgacggca ccgcctcgga
     9001 cggcgagcag gcgctctaca tgagcatgcc ctgcccctca ggttaatgat aggcttatat
     9061 cttcttgcaa caatggtagc gcgattgatg agataataga taaaaaaatc cactgtgaat
     9121 gtttttcaat gagtctacaa tagtgacttg acaggtcgta tagagtgaga cattagttat
     9181 ctctaaccaa ccctgctcgc tatacaggag agtgtgcaat gcagtcaatt caatagatat
     9241 gtaaaatgtt gatgtaaggg aaacaatcaa gaacagattt cagatcaaac tgaatgaact
     9301 cttgcacttt gacac
//
