LOCUS       sequence-432999-        8212 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express mKate-TEV_cutSite_YFP to the peroxisome with high expression
            TEV protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8212)
  AUTHORS   Baker JJ, Shi J, Wang S, Mujica EM, Bianco S, Capponi S, Dueber JE
  TITLE     ML-enhanced peroxisome capacity enables compartmentalization of 
            multienzyme pathway.
  JOURNAL   Nat Chem Biol. 2024 Oct 14. doi: 10.1038/s41589-024-01759-2.
  PUBMED    39402374
REFERENCE   2  (bases 1 to 8212)
  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: "Nat Chem 
            Biol. 2024 Oct 14. doi: 10.1038/s41589-024-01759-2."
COMMENT     Sequence Label: pJJB1463
FEATURES             Location/Qualifiers
     source          1..8212
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             732..1427
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     CDS             1434..1454
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1461..2171
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKL
                     ICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEGGNILGHKLEYNYNSHNVYITADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     terminator      2229..2416
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2284..2308)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        2500..3166
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     3036..3059
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     CDS             3176..3883
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     promoter        4306..4493
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             4494..5156
                     /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(5930..6745)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(5930..6745)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     6653..6672
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6737..6756
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6970..7558
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7459..7478
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 actggccgat aattgcagac gaacgaagag gatgtccaat atttttttta aggaataagg
       61 atacttcaag actagattcc cccctgcatt cccatcagaa ccgtaaacct tggcgctttc
      121 cttgggaagt attcaagaag tgccttgtcc ggtttctgtg gctcacaaac cagcgcgccc
      181 gatatggctt tcttttcact tatgaatgta ccagtacggg acaattagaa cgctcctgta
      241 acaatctctt tgcaaatgtg gggttacatt ctaaccatgt cacactgctg acgaaattca
      301 aagtaaaaaa aaatgggacc acgtcttgag aacgatagat tttctttatt ttacattgaa
      361 cagtcgttgt ctcagcgcgc tttatgtttt cattcatact tcatattata aaataacaaa
      421 agaagaattt catattcacg cccaagaaat caggctgctt tccaaatgca attgacactt
      481 cattagccat cacacaaaac tctttcttgc tggagcttct tttaaaaaag acctcagtac
      541 accaaacacg ttacccgacc tcgttatttt acgacaacta tgataaaatt ctgaagaaaa
      601 aataaaaaaa ttttcatact tcttgctttt atttaaacca ttgaatgatt tcttttgaac
      661 aaaactacct gtttcaccaa aggaaataga aagaaaaaat caattagaag aaaacaaaaa
      721 acaaaagatc tatggtgagc gagctgatta aggagaacat gcacatgaag ctgtacatgg
      781 agggcaccgt gaacaaccac cacttcaagt gcacatccga gggcgaaggc aagccctacg
      841 agggcaccca gaccatgaga atcaaggcgg tcgagggcgg ccctctcccc ttcgccttcg
      901 acatcctggc taccagcttc atgtacggca gcaaaacctt catcaaccac acccagggca
      961 tccccgactt ctttaagcag tccttccccg agggcttcac atgggagaga gtcaccacat
     1021 acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca
     1081 tctacaacgt caagatcaga ggggtgaact tcccatccaa cggccctgtg atgcagaaga
     1141 aaacactcgg ctgggaggcc tccaccgaaa ccctgtaccc cgctgacggc ggcctggaag
     1201 gcagagccga catggccctg aagctcgttg gcgggggcca cctgatctgc aacttgaaga
     1261 ccacatacag atccaagaaa cccgctaaga acctcaagat gcccggcgtc tactatgtgg
     1321 acagaagact ggaaagaatc aaggaggccg acaaagaaac ctacgtcgag cagcacgagg
     1381 tggctgtggc cagatactgc gacctcccta gcaaactggg gcacagagga tccgagaatt
     1441 tgtactttca atctggtagc tctaaaggtg aagaattatt cactggtgtt gtcccaattt
     1501 tggttgaatt agatggtgat gttaatggtc acaaattttc tgtctccggt gaaggtgaag
     1561 gtgatgctac ttacggtaaa ttgaccttaa aattgatttg tactactggt aaattgccag
     1621 ttccatggcc aaccttagtc actactttag gttatggttt gcaatgtttt gctagatacc
     1681 cagatcatat gaaacaacat gactttttca agtctgccat gccagaaggt tatgttcaag
     1741 aaagaactat ttttttcaaa gatgacggta actacaagac cagagctgaa gtcaagtttg
     1801 aaggtgatac cttagttaat agaatcgaat taaaaggtat tgattttaaa gaaggtggta
     1861 acattttagg tcacaaattg gaatacaact ataactctca caatgtttac atcactgctg
     1921 acaaacaaaa gaatggtatc aaagctaact tcaaaattag acacaacatt gaagatggtg
     1981 gtgttcaatt agctgaccat tatcaacaaa atactccaat tggtgatggt ccagtcttgt
     2041 taccagacaa ccattactta tcctatcaat ctgccttatc caaagatcca aacgaaaaga
     2101 gagatcacat ggtcttgtta gaatttgtta ctgctgctgg tattacccat ggtatggatg
     2161 aattgtacaa aggatccggt tcattgggaa gaggtagaag atccaaattg taaggatcct
     2221 aactcgaggc gaatttctta tgatttatga tttttattat taaataagtt ataaaaaaaa
     2281 taagtgtata caaattttaa agtgactctt aggttttaaa acgaaaattc ttattcttga
     2341 gtaactcttt cctgtaggtc aggttgcttt ctcaggtata gcatgaggtc gctcttattg
     2401 accacacctc taccggcatg ccgagcaaat gcctgcaaat cgctccccat ttcgctgcca
     2461 aaccagatgt caacacagct acaacgcagt tcgagtttat cattatcaat actgccattt
     2521 caaagaatac gtaaataatt aatagtagtg attttcctaa ctttatttag tcaaaaaatt
     2581 agccttttaa ttctgctgta acccgtacat gcccaaaata gggggcgggt tacacagaat
     2641 atataacatc gtaggtgtct gggtgaacag tttattcctg gcatccacta aatataatgg
     2701 agcccgcttt ttaagctggc atccagaaaa aaaaagaatc ccagcaccaa aatattgttt
     2761 tcttcaccaa ccatcagttc ataggtccat tctcttagcg caactacaga gaacaggggc
     2821 acaaacaggc aaaaaacggg cacaacctca atggagtgat gcaacctgcc tggagtaaat
     2881 gatgacacaa ggcaattgac ccacgcatgt atctatctca ttttcttaca ccttctatta
     2941 ccttctgctc tctctgattt ggaaaaagct gaaaaaaaag gttgaaacca gttccctgaa
     3001 attattcccc tacttgacta ataagtatat aaagacggta ggtattgatt gtaattctgt
     3061 aaatctattt cttaaacttc ttaaattcta cttttatagt tagtcttttt tttagtttta
     3121 aaacaccaag aacttagttt cgaataaaca cacataaaca aacaaaagat ctatgggaga
     3181 aagcttgttt aaggggccgc gtgattacaa cccgatatcg agcaccattt gtcatttgac
     3241 gaatgaatct gatgggcaca caacatcgtt gtatggtatt ggatttggtc ccttcatcat
     3301 tacaaacaag cacttgttta gaagaaataa tggaacactg ttggtccaat cactacatgg
     3361 tgtattcaag gtcaagaaca ccacgacttt gcaacaacac ctcattgatg ggagggacat
     3421 gataattatt cgcatgccta aggatttccc accatttcct caaaagctga aatttagaga
     3481 gccacaaagg gaagagcgca tatgtcttgt gacaaccaac ttccaaacta agagcatgtc
     3541 tagcatggtg tcagacacta gttgcacatt cccttcatct gatggcatat tctggaagca
     3601 ttggattcaa accaaggatg ggcagtgtgg cagtccatta gtatcaacta gagatgggtt
     3661 cattgttggt atacactcag catcgaattt caccaacaca aacaattatt tcacaagcgt
     3721 gccgaaaaac ttcatggaat tgttgacaaa tcaggaggcg cagcagtggg ttagtggttg
     3781 gcgattaaat gctgactcag tattgtgggg gggccataaa gttttcatgg tgaaacctga
     3841 agagcctttt cagccagtta aggaagcgac tcaactcatg aatcgtcgtc gccgtcgcgg
     3901 atcctaactc gagattgaat tgaattgaaa tcgatagatc aatttttttc ttttctcttt
     3961 ccccatcctt tacgctaaaa taatagttta ttttattttt tgaatatttt ttatttatat
     4021 acgtatatat agactattat ttatctttta atgattatta agatttttat taaaaaaaaa
     4081 ttcgctcctc ttttaatgcc tttatgcagt ttttttttcc cattcgatat ttctatgtgc
     4141 tggatgcaca cactggctta agatgacaac gactagagat ctatgtgagg atcctaactc
     4201 gagatcgagc tgagcagtta cagagatgtt acgaaccact agtgcactgc agtacaaaca
     4261 cagtcctttc ccgcaatttt ctttttctat tactcttggc ctcctctagt acactctata
     4321 tttttttatg cctcggtaat gattttcatt tttttttttc cacctagcgg atgactcttt
     4381 ttttttctta gcgattggca ttatcacata atgaattata cattatataa agtaatgtga
     4441 tttcttcgaa gaatatacta aaaaatgagc aggcaagata aacgaaggca aagatgacag
     4501 agcagaaagc cctagtaaag cgtattacaa atgaaaccaa gattcagatt gcgatctctt
     4561 taaagggtgg tcccctagcg atagagcact cgatcttccc agaaaaagag gcagaagcag
     4621 tagcagaaca ggccacacaa tcgcaagtga ttaacgtcca cacaggtata gggtttctgg
     4681 accatatgat acatgctctg gccaagcatt ccggctggtc gctaatcgtt gagtgcattg
     4741 gtgacttaca catagacgac catcacacca ctgaagactg cgggattgct ctcggtcaag
     4801 cttttaaaga ggccctaggg gccgtgcgtg gagtaaaaag gtttggatca ggatttgcgc
     4861 ctttggatga ggcactttcc agagcggtgg ttgatctttc gaacaggccg tacgcagttg
     4921 tcgaacttgg tttgcaaagg gagaaagtag gtgatctctc ttgcgagatg atcccgcatt
     4981 ttcttgaaag ctttgcagag gctagcagaa ttaccctcca cgttgattgt ctgcgaggca
     5041 agaatgatca tcaccgtagt gagagtgcgt tcaaggctct tgcggttgcc ataagagaag
     5101 ccacctcgcc caatggtacc aacgatgttc cctccaccaa aggtgttctt atgtagtgac
     5161 accgattatt taaagctgct gcatacgata tatatacatg tgtatatatg tatacctatg
     5221 aatgtcagta agtatgtata cgaacagtat gatactgaag atgacaaggt aatgcatcat
     5281 tctatacgtg tcattctgaa cgaggcgcgc tttccttttt tctttttgct ttttcttttt
     5341 ttttctcttg aactcgacgg atcatagagt cgcacagtgt ttcaagatgc acatgtgtat
     5401 atatgtatac ctatgaatgt cagtaagtat gtatacgaac agtatgatac tgaagatgac
     5461 aaggtaatgc atcattctat acgtgtcatt ctgaacgagg cgcgctttcc ttttttcttt
     5521 ttgctttttc tttttttttc tcttgaacac gagaaaaaaa atataaaaga gatggaggaa
     5581 cgggaaaaag ttagttgtgg tgataggtgg caagtggtat tccgtaagaa caacaagaaa
     5641 agcatttcat attatggctg aactgagcga acaagtgcaa aatttaagca tcaacgacaa
     5701 caacgagaat ggttatgttc ctcctcactt aagaggaaaa ccaagaagtg ccagaaataa
     5761 cagtagcaac tacaataaca acaacggcgg ctacaacggt ggccgtggcg gtggcagctt
     5821 ctttagcaac aaccgtcgtg gtggttacgg caacggtggt ttcttcggtg gaaacaacgg
     5881 tggcagcaga ccgagcggcc gcgtgttaca accaattaac caattctgat tagaaaaact
     5941 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     6001 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     6061 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     6121 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     6181 agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     6241 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     6301 ggcgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     6361 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     6421 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     6481 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     6541 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     6601 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     6661 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctggagcaag
     6721 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     6781 gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag
     6841 acacaacgtg gctttgttga ataaatcgaa cttttgctga gttgaaggat cagtcatgac
     6901 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6961 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     7021 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     7081 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     7141 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     7201 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     7261 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     7321 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     7381 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     7441 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     7501 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7561 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     7621 ctttcctgcg ttatcccctg attctgtgga taaccgtgcg gccgccaatg taaccttcag
     7681 ttcatcgact atctgaagaa atttactagg aatagtgcca tggtacagca accgagaatg
     7741 gcaatttcta ctcgggttca gcaacgctgc ataaacgctg ttggtgccgt agacatattc
     7801 gaagatagga ttatcattca taagtttcag agcaatgtcc ttattctgga acttggattt
     7861 atggctcttt tggtttaatt tcgcctgatt cttgatctcc tttagcttct cgacgtgggc
     7921 ctttttcttg ccatatgggt ccgctgcacg gtcctgttcc ctagcatgta cgtgagcgta
     7981 tttcctttta aaccacgacg ctttgtcctc attcaacgtt tcccattgtt tttttctact
     8041 attgctttgc tgtgggaaaa acttatcgaa agatgacgac tttttcttaa ttctcgtttt
     8101 aagagcttgg tgagcgctag gagtcactgc caggtatcgt ttgaacacgg cattagtcag
     8161 ggaagtcatc agacgcatga acttgaagcc cctgaattcg catctagact ga
//
