LOCUS       sequence-432996-        8232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express mKate-TEV_cutSite_YFP to the peroxisome with low expression 
            TEV protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8232)
  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 8232)
  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: pJJB1462
FEATURES             Location/Qualifiers
     source          1..8232
                     /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"
     CDS             3196..3903
                     /codon_start=1
                     /product="tobacco etch virus protease (Kapust et al., 
                     2001)"
                     /label=TEV protease
                     /translation="GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNK
                     HLFRRNNGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQ
                     REERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFI
                     VGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPE
                     EPFQPVKEATQLMN"
     promoter        4326..4513
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             4514..5176
                     /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(5950..6765)
                     /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(5950..6765)
                     /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     6673..6692
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6757..6776
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6990..7578
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7479..7498
                     /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 acaacggtgt tgttatccga tacaaccgga tatttttctt
     2521 ttaatgagtc taaaccgtga tagcttcagg ttaatacaat caaaaaaagc tcaaatattc
     2581 ttttaatgcc gcgttcacag attccaattg aatacaacta ggtagttcat tatatgaagc
     2641 ctttgctact atttttcact atagtctgcc ttcaccttaa tgcagacatc cacatatttt
     2701 aatcacttta aaataaaaag gaagatatat tagaagctat gatccaatct gtaagccaga
     2761 ttaaaattca cgaactcttc tttcatttga attgaatgct ttgagttggg gtagattatc
     2821 gcaaattact catcacattt attgactacg aacttgctga tgtccttttt ttatttatat
     2881 ttttcttcag tgaagcgatt ttttttttac acagaccaag acggaaaaaa gtagctaagg
     2941 aagaaaacaa aatcatgaaa aaaatgtgaa gtgatcatgc acatcgcatc aacttaaaca
     3001 ttggcttaga gatatataga gttagagttt acggcaacct ttaagcacca ataccttttg
     3061 gcatagtcta aagacctggt tcttaatttt aaacaaattt aactaaagat ttccctatca
     3121 aagaagtaac gagttgacag attttctcaa aataaatcga tactgcattt ctaggcatat
     3181 ccagcgagat ctatgggaga aagcttgttt aaggggccgc gtgattacaa cccgatatcg
     3241 agcaccattt gtcatttgac gaatgaatct gatgggcaca caacatcgtt gtatggtatt
     3301 ggatttggtc ccttcatcat tacaaacaag cacttgttta gaagaaataa tggaacactg
     3361 ttggtccaat cactacatgg tgtattcaag gtcaagaaca ccacgacttt gcaacaacac
     3421 ctcattgatg ggagggacat gataattatt cgcatgccta aggatttccc accatttcct
     3481 caaaagctga aatttagaga gccacaaagg gaagagcgca tatgtcttgt gacaaccaac
     3541 ttccaaacta agagcatgtc tagcatggtg tcagacacta gttgcacatt cccttcatct
     3601 gatggcatat tctggaagca ttggattcaa accaaggatg ggcagtgtgg cagtccatta
     3661 gtatcaacta gagatgggtt cattgttggt atacactcag catcgaattt caccaacaca
     3721 aacaattatt tcacaagcgt gccgaaaaac ttcatggaat tgttgacaaa tcaggaggcg
     3781 cagcagtggg ttagtggttg gcgattaaat gctgactcag tattgtgggg gggccataaa
     3841 gttttcatgg tgaaacctga agagcctttt cagccagtta aggaagcgac tcaactcatg
     3901 aatcgtcgtc gccgtcgcgg atcctaactc gagattgaat tgaattgaaa tcgatagatc
     3961 aatttttttc ttttctcttt ccccatcctt tacgctaaaa taatagttta ttttattttt
     4021 tgaatatttt ttatttatat acgtatatat agactattat ttatctttta atgattatta
     4081 agatttttat taaaaaaaaa ttcgctcctc ttttaatgcc tttatgcagt ttttttttcc
     4141 cattcgatat ttctatgtgc tggatgcaca cactggctta agatgacaac gactagagat
     4201 ctatgtgagg atcctaactc gagatcgagc tgagcagtta cagagatgtt acgaaccact
     4261 agtgcactgc agtacaaaca cagtcctttc ccgcaatttt ctttttctat tactcttggc
     4321 ctcctctagt acactctata tttttttatg cctcggtaat gattttcatt tttttttttc
     4381 cacctagcgg atgactcttt ttttttctta gcgattggca ttatcacata atgaattata
     4441 cattatataa agtaatgtga tttcttcgaa gaatatacta aaaaatgagc aggcaagata
     4501 aacgaaggca aagatgacag agcagaaagc cctagtaaag cgtattacaa atgaaaccaa
     4561 gattcagatt gcgatctctt taaagggtgg tcccctagcg atagagcact cgatcttccc
     4621 agaaaaagag gcagaagcag tagcagaaca ggccacacaa tcgcaagtga ttaacgtcca
     4681 cacaggtata gggtttctgg accatatgat acatgctctg gccaagcatt ccggctggtc
     4741 gctaatcgtt gagtgcattg gtgacttaca catagacgac catcacacca ctgaagactg
     4801 cgggattgct ctcggtcaag cttttaaaga ggccctaggg gccgtgcgtg gagtaaaaag
     4861 gtttggatca ggatttgcgc ctttggatga ggcactttcc agagcggtgg ttgatctttc
     4921 gaacaggccg tacgcagttg tcgaacttgg tttgcaaagg gagaaagtag gtgatctctc
     4981 ttgcgagatg atcccgcatt ttcttgaaag ctttgcagag gctagcagaa ttaccctcca
     5041 cgttgattgt ctgcgaggca agaatgatca tcaccgtagt gagagtgcgt tcaaggctct
     5101 tgcggttgcc ataagagaag ccacctcgcc caatggtacc aacgatgttc cctccaccaa
     5161 aggtgttctt atgtagtgac accgattatt taaagctgct gcatacgata tatatacatg
     5221 tgtatatatg tatacctatg aatgtcagta agtatgtata cgaacagtat gatactgaag
     5281 atgacaaggt aatgcatcat tctatacgtg tcattctgaa cgaggcgcgc tttccttttt
     5341 tctttttgct ttttcttttt ttttctcttg aactcgacgg atcatagagt cgcacagtgt
     5401 ttcaagatgc acatgtgtat atatgtatac ctatgaatgt cagtaagtat gtatacgaac
     5461 agtatgatac tgaagatgac aaggtaatgc atcattctat acgtgtcatt ctgaacgagg
     5521 cgcgctttcc ttttttcttt ttgctttttc tttttttttc tcttgaacac gagaaaaaaa
     5581 atataaaaga gatggaggaa cgggaaaaag ttagttgtgg tgataggtgg caagtggtat
     5641 tccgtaagaa caacaagaaa agcatttcat attatggctg aactgagcga acaagtgcaa
     5701 aatttaagca tcaacgacaa caacgagaat ggttatgttc ctcctcactt aagaggaaaa
     5761 ccaagaagtg ccagaaataa cagtagcaac tacaataaca acaacggcgg ctacaacggt
     5821 ggccgtggcg gtggcagctt ctttagcaac aaccgtcgtg gtggttacgg caacggtggt
     5881 ttcttcggtg gaaacaacgg tggcagcaga ccgagcggcc gcgtgttaca accaattaac
     5941 caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     6001 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     6061 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     6121 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     6181 agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc
     6241 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     6301 tcgtgattgc gcctgagcga ggcgaaatac gcgatcgctg ttaaaaggac aattacaaac
     6361 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     6421 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     6481 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     6541 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     6601 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     6661 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     6721 taatcgcggc ctggagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     6781 actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat
     6841 gtaacatcag agattttgag acacaacgtg gctttgttga ataaatcgaa cttttgctga
     6901 gttgaaggat cagtcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6961 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     7021 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     7081 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     7141 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     7201 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7261 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7321 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7381 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7441 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7501 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7561 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7621 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtgcg
     7681 gccgccaatg taaccttcag ttcatcgact atctgaagaa atttactagg aatagtgcca
     7741 tggtacagca accgagaatg gcaatttcta ctcgggttca gcaacgctgc ataaacgctg
     7801 ttggtgccgt agacatattc gaagatagga ttatcattca taagtttcag agcaatgtcc
     7861 ttattctgga acttggattt atggctcttt tggtttaatt tcgcctgatt cttgatctcc
     7921 tttagcttct cgacgtgggc ctttttcttg ccatatgggt ccgctgcacg gtcctgttcc
     7981 ctagcatgta cgtgagcgta tttcctttta aaccacgacg ctttgtcctc attcaacgtt
     8041 tcccattgtt tttttctact attgctttgc tgtgggaaaa acttatcgaa agatgacgac
     8101 tttttcttaa ttctcgtttt aagagcttgg tgagcgctag gagtcactgc caggtatcgt
     8161 ttgaacacgg cattagtcag ggaagtcatc agacgcatga acttgaagcc cctgaattcg
     8221 catctagact ga
//
