LOCUS       sequence-318011-        5329 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High-copy expression vector for K. marxianus, leucine selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5329)
  AUTHORS   Rajkumar AS, Morrissey JP
  TITLE     Rational engineering of Kluyveromyces marxianus to create a chassis 
            for the production of aromatic products.
  JOURNAL   Microb Cell Fact. 2020 Nov 11;19(1):207. doi: 
            10.1186/s12934-020-01461-7.
  PUBMED    33176787
REFERENCE   2  (bases 1 to 5329)
  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: "Microb Cell
            Fact."; date: "2020-11-11"; volume: "19(1):207. doi"; pages: " 
            10.1186/s12934-020-01461-7"
COMMENT     Sequence Label: pMTU-DO-LEU-C4
FEATURES             Location/Qualifiers
     source          1..5329
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      294..365
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      381..408
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        860..1267
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             1268..2362
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     CDS             complement(2812..3627)
                     /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(2812..3627)
                     /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     3535..3554
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3619..3638
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3852..4440
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4341..4360
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             4880..4891
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(4898..5329,1..282)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
ORIGIN
        1 tttaacagcc acaatgttta catcaccgcc gataaacaaa aaaatggcat taaagcgaat
       61 tttaaaattc gccacaacgt ggaggatggc agcgtgcagc tggctgatca ctaccagcaa
      121 aacactccaa tcggtgatgg tcctgttctg ctgccagaca atcactatct gagcacgcaa
      181 agcgttctgt ctaaagatcc gaacgagaaa cgcgatcata tggttctgct ggagttcgta
      241 accgcagcgg gcatcacgca tggtatggat gaactgtaca aatgaccagg catcaaataa
      301 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
      361 ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tataggtctc
      421 agctggaaat ctgctcgtca gtggtgctca cactgacgaa tcatgtacag atcataccga
      481 tgactgcctg gcgactcaca actaagcaag acagccggaa ccagcgccgg cgaacaccac
      541 tgcatatatg gcatatcaca acagtccacg tctcaagcag ttacagagat gttacgaacc
      601 actagtgcac tgcagtacat cgaggagaac ttctagtata tctacatacc taatattatt
      661 gccttattaa aaatggaatc ccaacaatta catcaaaatc cacattctct tcaaaatcaa
      721 ttgtcctgta cttccttgtt catgtgtgtt caaaaacgtt atatttatag gataattata
      781 ctctatttct caacaagtaa ttggttgttt ggccgagcgg tctaaggcgc ctgattcaag
      841 aaatatcttg accgcagtta actgtgggaa tactcaggta tcgtaagatg caagagttcg
      901 aatctcttag caaccattat ttttttcctc aacataacga gaacacacag gggcgctatc
      961 gcacagaatc aaattcgatg actggaaatt ttttgttaat ttcagaggtc gcctgacgca
     1021 tatacctttt tcaactgaaa aattgggaga aaaaggaaag gtgagagcgc cggaaccggc
     1081 ttttcatata gaatagagaa gcgttcatga ctaaatgctt gcatcacaat acttgaagtt
     1141 gacaatatta tttaaggacc tattgttttt tccaataggt ggttagcaat cgtcttactt
     1201 tctaactttt cttacctttt acatttcagc aatatatata tatatatttc aaggatatac
     1261 cattctaatg tctgccccta agaagatcgt cgttttgcca ggtgaccacg ttggtcaaga
     1321 aatcacagcc gaagccatta aggttcttaa agctatttct gatgttcgtt ccaatgtcaa
     1381 gttcgatttc gaaaatcatt taattggtgg tgctgctatc gatgctacag gtgttccact
     1441 tccagatgag gcgctggaag cctccaagaa ggctgatgcc gttttgttag gtgctgtggg
     1501 tggtcctaaa tggggaaccg gtagtgttag acctgaacaa ggtttactaa aaatccgtaa
     1561 agaacttcaa ttgtacgcca acttaagacc atgtaacttt gcatccgact ctcttttaga
     1621 cttatctcca atcaagccac aatttgctaa aggtactgac ttcgttgttg tcagagaatt
     1681 agtgggaggt atttactttg gtaagagaaa ggaggacgat ggtgatggtg tcgcttggga
     1741 tagtgaacaa tacaccgttc cagaagtgca aagaatcaca agaatggccg ctttcatggc
     1801 cctacaacat gagccaccat tgcctatttg gtccttggat aaagctaatg ttttggcctc
     1861 ttcaagatta tggagaaaaa ctgtggagga aaccatcaag aacgaatttc ctacattgaa
     1921 ggttcaacat caattgattg attctgccgc catgatccta gttaagaacc caacccacct
     1981 aaatggtatt ataatcacca gcaacatgtt tggtgatatc atctccgatg aagcctccgt
     2041 tatcccaggt tccttgggtt tgttgccatc tgcgtccttg gcctctttgc cagacaagaa
     2101 caccgcattt ggtttgtacg aaccatgcca cggttctgct ccagatttgc caaagaataa
     2161 ggtcaaccct atcgccacta tcttgtctgc tgcaatgatg ttgaaattgt cattgaactt
     2221 gcctgaagaa ggtaaggcca ttgaagatgc agttaaaaag gttttggatg caggtatcag
     2281 aactggtgat ttaggtggtt ccaacagtac caccgaagtc ggtgatgctg tcgccgaaga
     2341 agttaagaaa atccttgctt aacctggagg acccttctct ttagactatt ctactcttat
     2401 gcacgtaaaa aattctagga aatatgtatt aactaggagt aaaataaccg gctagtggca
     2461 ttcatatagc cgtctgttta catctacatc acacatttcg agtgtatatc tcgcaacgtt
     2521 ggcgttaaat aggcaggagt caatactttc taacattgac cattttgcat cattttgtta
     2581 cggaaaagtg ctttccaatc atatataatt aaaactagcg gatcacatta aaaagttcat
     2641 ccaaagtgaa aaaacattct aaatttagta gacagcaatt gttttaaaag ggtgttccta
     2701 ttttcatata acggagagag ttaagcttcc tgtatgcaat tagcaaatat tttcactgta
     2761 gagttctggg acccgagcgg ccgcgtgtta caaccaatta accaattctg attagaaaaa
     2821 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     2881 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     2941 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     3001 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     3061 tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     3121 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     3181 gaggcgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     3241 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     3301 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     3361 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     3421 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     3481 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     3541 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctggagca
     3601 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     3661 cagttttatt gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg
     3721 agacacaacg tggctttgtt gaataaatcg aacttttgct gagttgaagg atcagtcatg
     3781 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3841 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3901 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3961 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     4021 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4081 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4141 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4201 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4261 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4321 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4381 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4441 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4501 ttctttcctg cgttatcccc tgattctgtg gataaccgtg cggccgcccc tgaattcgca
     4561 tctagactga tgagacgtgg tagagccaca aacagccggt acaagcaacg atctccagga
     4621 ccatctgaat catgcgcgga tgacacgaac tcacgacggc gatcacagac attaacccac
     4681 agtacagaca ctgcgacaac gtggcaattc gtcgcaatac aacgtgagac cgaaagtgaa
     4741 acgtgatttc atgcgtcatt ttgaacattt tgtaaatctt atttaataat gtgtgcggca
     4801 attcacattt aatttatgaa tgttttctta acatcgcggc aactcaagaa acggcaggtt
     4861 cggatcttag ctactagaga aagaggagaa atactagatg cgtaaaggcg aagagctgtt
     4921 cactggtgtc gtccctattc tggtggaact ggatggtgat gtcaacggtc ataagttttc
     4981 cgtgcgtggc gagggtgaag gtgacgcaac taatggtaaa ctgacgctga agttcatctg
     5041 tactactggt aaactgccgg ttccttggcc gactctggta acgacgctga cttatggtgt
     5101 tcagtgcttt gctcgttatc cggaccatat gaagcagcat gacttcttca agtccgccat
     5161 gccggaaggc tatgtgcagg aacgcacgat ttcctttaag gatgacggca cgtacaaaac
     5221 gcgtgcggaa gtgaaatttg aaggcgatac cctggtaaac cgcattgagc tgaaaggcat
     5281 tgactttaaa gaggacggca atatcctggg ccataagctg gaatacaat
//
