LOCUS       sequence-310508-        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"
     RBS             333..344
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             351..1064
                     /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"
     terminator      1076..1147
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1163..1190
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        1642..2049
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2050..3144
                     /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(3594..4409)
                     /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(3594..4409)
                     /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     4317..4336
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4401..4420
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4634..5222
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5123..5142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccctgaattc gcatctagac tgatgagacg tggtagagcc acaaacagcc ggtacaagca
       61 acgatctcca ggaccatctg aatcatgcgc ggatgacacg aactcacgac ggcgatcaca
      121 gacattaacc cacagtacag acactgcgac aacgtggcaa ttcgtcgcaa tacaacgtga
      181 gaccgaaagt gaaacgtgat ttcatgcgtc attttgaaca ttttgtaaat cttatttaat
      241 aatgtgtgcg gcaattcaca tttaatttat gaatgttttc ttaacatcgc ggcaactcaa
      301 gaaacggcag gttcggatct tagctactag agaaagagga gaaatactag atgcgtaaag
      361 gcgaagagct gttcactggt gtcgtcccta ttctggtgga actggatggt gatgtcaacg
      421 gtcataagtt ttccgtgcgt ggcgagggtg aaggtgacgc aactaatggt aaactgacgc
      481 tgaagttcat ctgtactact ggtaaactgc cggttccttg gccgactctg gtaacgacgc
      541 tgacttatgg tgttcagtgc tttgctcgtt atccggacca tatgaagcag catgacttct
      601 tcaagtccgc catgccggaa ggctatgtgc aggaacgcac gatttccttt aaggatgacg
      661 gcacgtacaa aacgcgtgcg gaagtgaaat ttgaaggcga taccctggta aaccgcattg
      721 agctgaaagg cattgacttt aaagaggacg gcaatatcct gggccataag ctggaataca
      781 attttaacag ccacaatgtt tacatcaccg ccgataaaca aaaaaatggc attaaagcga
      841 attttaaaat tcgccacaac gtggaggatg gcagcgtgca gctggctgat cactaccagc
      901 aaaacactcc aatcggtgat ggtcctgttc tgctgccaga caatcactat ctgagcacgc
      961 aaagcgttct gtctaaagat ccgaacgaga aacgcgatca tatggttctg ctggagttcg
     1021 taaccgcagc gggcatcacg catggtatgg atgaactgta caaatgacca ggcatcaaat
     1081 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     1141 cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg tttataggtc
     1201 tcagctggaa atctgctcgt cagtggtgct cacactgacg aatcatgtac agatcatacc
     1261 gatgactgcc tggcgactca caactaagca agacagccgg aaccagcgcc ggcgaacacc
     1321 actgcatata tggcatatca caacagtcca cgtctcaagc agttacagag atgttacgaa
     1381 ccactagtgc actgcagtac atcgaggaga acttctagta tatctacata cctaatatta
     1441 ttgccttatt aaaaatggaa tcccaacaat tacatcaaaa tccacattct cttcaaaatc
     1501 aattgtcctg tacttccttg ttcatgtgtg ttcaaaaacg ttatatttat aggataatta
     1561 tactctattt ctcaacaagt aattggttgt ttggccgagc ggtctaaggc gcctgattca
     1621 agaaatatct tgaccgcagt taactgtggg aatactcagg tatcgtaaga tgcaagagtt
     1681 cgaatctctt agcaaccatt atttttttcc tcaacataac gagaacacac aggggcgcta
     1741 tcgcacagaa tcaaattcga tgactggaaa ttttttgtta atttcagagg tcgcctgacg
     1801 catatacctt tttcaactga aaaattggga gaaaaaggaa aggtgagagc gccggaaccg
     1861 gcttttcata tagaatagag aagcgttcat gactaaatgc ttgcatcaca atacttgaag
     1921 ttgacaatat tatttaagga cctattgttt tttccaatag gtggttagca atcgtcttac
     1981 tttctaactt ttcttacctt ttacatttca gcaatatata tatatatatt tcaaggatat
     2041 accattctaa tgtctgcccc taagaagatc gtcgttttgc caggtgacca cgttggtcaa
     2101 gaaatcacag ccgaagccat taaggttctt aaagctattt ctgatgttcg ttccaatgtc
     2161 aagttcgatt tcgaaaatca tttaattggt ggtgctgcta tcgatgctac aggtgttcca
     2221 cttccagatg aggcgctgga agcctccaag aaggctgatg ccgttttgtt aggtgctgtg
     2281 ggtggtccta aatggggaac cggtagtgtt agacctgaac aaggtttact aaaaatccgt
     2341 aaagaacttc aattgtacgc caacttaaga ccatgtaact ttgcatccga ctctctttta
     2401 gacttatctc caatcaagcc acaatttgct aaaggtactg acttcgttgt tgtcagagaa
     2461 ttagtgggag gtatttactt tggtaagaga aaggaggacg atggtgatgg tgtcgcttgg
     2521 gatagtgaac aatacaccgt tccagaagtg caaagaatca caagaatggc cgctttcatg
     2581 gccctacaac atgagccacc attgcctatt tggtccttgg ataaagctaa tgttttggcc
     2641 tcttcaagat tatggagaaa aactgtggag gaaaccatca agaacgaatt tcctacattg
     2701 aaggttcaac atcaattgat tgattctgcc gccatgatcc tagttaagaa cccaacccac
     2761 ctaaatggta ttataatcac cagcaacatg tttggtgata tcatctccga tgaagcctcc
     2821 gttatcccag gttccttggg tttgttgcca tctgcgtcct tggcctcttt gccagacaag
     2881 aacaccgcat ttggtttgta cgaaccatgc cacggttctg ctccagattt gccaaagaat
     2941 aaggtcaacc ctatcgccac tatcttgtct gctgcaatga tgttgaaatt gtcattgaac
     3001 ttgcctgaag aaggtaaggc cattgaagat gcagttaaaa aggttttgga tgcaggtatc
     3061 agaactggtg atttaggtgg ttccaacagt accaccgaag tcggtgatgc tgtcgccgaa
     3121 gaagttaaga aaatccttgc ttaacctgga ggacccttct ctttagacta ttctactctt
     3181 atgcacgtaa aaaattctag gaaatatgta ttaactagga gtaaaataac cggctagtgg
     3241 cattcatata gccgtctgtt tacatctaca tcacacattt cgagtgtata tctcgcaacg
     3301 ttggcgttaa ataggcagga gtcaatactt tctaacattg accattttgc atcattttgt
     3361 tacggaaaag tgctttccaa tcatatataa ttaaaactag cggatcacat taaaaagttc
     3421 atccaaagtg aaaaaacatt ctaaatttag tagacagcaa ttgttttaaa agggtgttcc
     3481 tattttcata taacggagag agttaagctt cctgtataca attagcaaat attttcacta
     3541 tagagacctg ggacccgagc ggccgcgtgt tacaaccaat taaccaattc tgattagaaa
     3601 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     3661 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     3721 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     3781 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     3841 ggtgagaatg gcaaaagctt atgcatttct ttccagactt gttcaacagg ccagccatta
     3901 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     3961 gcgaggcgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     4021 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     4081 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     4141 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     4201 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     4261 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     4321 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctggag
     4381 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     4441 gacagtttta ttgttcatga tgatatattt ttatcttgtg caatgtaaca tcagagattt
     4501 tgagacacaa cgtggctttg ttgaataaat cgaacttttg ctgagttgaa ggatcagtca
     4561 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4621 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4681 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4741 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     4801 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4861 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4921 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4981 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5041 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5101 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5161 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5221 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5281 tgttctttcc tgcgttatcc cctgattctg tggataaccg tgcggccgc
//
