LOCUS       sequence-317069-        5488 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low-copy expression vector for K. marxianus, leucine selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5488)
  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 5488)
  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
FEATURES             Location/Qualifiers
     source          1..5488
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             329..340
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             347..1060
                     /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      1072..1143
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1159..1186
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        1638..2045
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2046..3140
                     /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(3749..4564)
                     /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(3749..4564)
                     /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     4472..4491
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4556..4575
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4789..5377
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5278..5297
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaattcgcat ctagactgat gagacgtggt agagccacaa acagccggta caagcaacga
       61 tctccaggac catctgaatc atgcgcggat gacacgaact cacgacggcg atcacagaca
      121 ttaacccaca gtacagacac tgcgacaacg tggcaattcg tcgcaataca acgtgagacc
      181 gaaagtgaaa cgtgatttca tgcgtcattt tgaacatttt gtaaatctta tttaataatg
      241 tgtgcggcaa ttcacattta atttatgaat gttttcttaa catcgcggca actcaagaaa
      301 cggcaggttc ggatcttagc tactagagaa agaggagaaa tactagatgc gtaaaggcga
      361 agagctgttc actggtgtcg tccctattct ggtggaactg gatggtgatg tcaacggtca
      421 taagttttcc gtgcgtggcg agggtgaagg tgacgcaact aatggtaaac tgacgctgaa
      481 gttcatctgt actactggta aactgccggt tccttggccg actctggtaa cgacgctgac
      541 ttatggtgtt cagtgctttg ctcgttatcc ggaccatatg aagcagcatg acttcttcaa
      601 gtccgccatg ccggaaggct atgtgcagga acgcacgatt tcctttaagg atgacggcac
      661 gtacaaaacg cgtgcggaag tgaaatttga aggcgatacc ctggtaaacc gcattgagct
      721 gaaaggcatt gactttaaag aggacggcaa tatcctgggc cataagctgg aatacaattt
      781 taacagccac aatgtttaca tcaccgccga taaacaaaaa aatggcatta aagcgaattt
      841 taaaattcgc cacaacgtgg aggatggcag cgtgcagctg gctgatcact accagcaaaa
      901 cactccaatc ggtgatggtc ctgttctgct gccagacaat cactatctga gcacgcaaag
      961 cgttctgtct aaagatccga acgagaaacg cgatcatatg gttctgctgg agttcgtaac
     1021 cgcagcgggc atcacgcatg gtatggatga actgtacaaa tgaccaggca tcaaataaaa
     1081 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     1141 ctctactaga gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta taggtctcag
     1201 ctggaaatct gctcgtcagt ggtgctcaca ctgacgaatc atgtacagat cataccgatg
     1261 actgcctggc gactcacaac taagcaagac agccggaacc agcgccggcg aacaccactg
     1321 catatatggc atatcacaac agtccacgtc tcaagcagtt acagagatgt tacgaaccac
     1381 tagtgcactg cagtacatcg aggagaactt ctagtatatc tacataccta atattattgc
     1441 cttattaaaa atggaatccc aacaattaca tcaaaatcca cattctcttc aaaatcaatt
     1501 gtcctgtact tccttgttca tgtgtgttca aaaacgttat atttatagga taattatact
     1561 ctatttctca acaagtaatt ggttgtttgg ccgagcggtc taaggcgcct gattcaagaa
     1621 atatcttgac cgcagttaac tgtgggaata ctcaggtatc gtaagatgca agagttcgaa
     1681 tctcttagca accattattt ttttcctcaa cataacgaga acacacaggg gcgctatcgc
     1741 acagaatcaa attcgatgac tggaaatttt ttgttaattt cagaggtcgc ctgacgcata
     1801 tacctttttc aactgaaaaa ttgggagaaa aaggaaaggt gagagcgccg gaaccggctt
     1861 ttcatataga atagagaagc gttcatgact aaatgcttgc atcacaatac ttgaagttga
     1921 caatattatt taaggaccta ttgttttttc caataggtgg ttagcaatcg tcttactttc
     1981 taacttttct taccttttac atttcagcaa tatatatata tatatttcaa ggatatacca
     2041 ttctaatgtc tgcccctaag aagatcgtcg ttttgccagg tgaccacgtt ggtcaagaaa
     2101 tcacagccga agccattaag gttcttaaag ctatttctga tgttcgttcc aatgtcaagt
     2161 tcgatttcga aaatcattta attggtggtg ctgctatcga tgctacaggt gttccacttc
     2221 cagatgaggc gctggaagcc tccaagaagg ctgatgccgt tttgttaggt gctgtgggtg
     2281 gtcctaaatg gggaaccggt agtgttagac ctgaacaagg tttactaaaa atccgtaaag
     2341 aacttcaatt gtacgccaac ttaagaccat gtaactttgc atccgactct cttttagact
     2401 tatctccaat caagccacaa tttgctaaag gtactgactt cgttgttgtc agagaattag
     2461 tgggaggtat ttactttggt aagagaaagg aggacgatgg tgatggtgtc gcttgggata
     2521 gtgaacaata caccgttcca gaagtgcaaa gaatcacaag aatggccgct ttcatggccc
     2581 tacaacatga gccaccattg cctatttggt ccttggataa agctaatgtt ttggcctctt
     2641 caagattatg gagaaaaact gtggaggaaa ccatcaagaa cgaatttcct acattgaagg
     2701 ttcaacatca attgattgat tctgccgcca tgatcctagt taagaaccca acccacctaa
     2761 atggtattat aatcaccagc aacatgtttg gtgatatcat ctccgatgaa gcctccgtta
     2821 tcccaggttc cttgggtttg ttgccatctg cgtccttggc ctctttgcca gacaagaaca
     2881 ccgcatttgg tttgtacgaa ccatgccacg gttctgctcc agatttgcca aagaataagg
     2941 tcaaccctat cgccactatc ttgtctgctg caatgatgtt gaaattgtca ttgaacttgc
     3001 ctgaagaagg taaggccatt gaagatgcag ttaaaaaggt tttggatgca ggtatcagaa
     3061 ctggtgattt aggtggttcc aacagtacca ccgaagtcgg tgatgctgtc gccgaagaag
     3121 ttaagaaaat ccttgcttaa cctggaggac ccttctcttt agactattct actcttatgc
     3181 acgtaaaaaa ttctaggaaa tatgtattaa ctaggagtaa aataaccggc tagtggcatt
     3241 catatagccg tctgtttaca tctacatcac acatttcgag tgtatatctc gcaacgttgg
     3301 cgttaaatag gcaggagttt ctacgcaaga cttcttgaag tgaaaaccaa ctttcagtct
     3361 tcaaactaaa aatgaaaatc agtgggctaa attcagcata atacctgtgg atataattac
     3421 atacaaaaat ataattaaaa atatacgtta aaaataattt atttttgtaa aaactataaa
     3481 atatatttta ctttcggaac aactttttaa cttataattt tgttttaaat aaaaacattg
     3541 tatttaaaaa taataaatta ttaaataaaa ttttaaagta tgtttattta aaatatagaa
     3601 tactattaaa attactttaa actttaaaat aaaaaaataa taaattatat agttttaaaa
     3661 ttaataaatt ttatacacgt gaccagatca taatagtttt ctttccttgc cgagcggccg
     3721 cgtgttacaa ccaattaacc aattctgatt agaaaaactc atcgagcatc aaatgaaact
     3781 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     3841 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     3901 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     3961 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agcttatgca
     4021 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     4081 caaccaaacc gttattcatt cgtgattgcg cctgagcgag gcgaaatacg cgatcgctgt
     4141 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     4201 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     4261 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     4321 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     4381 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     4441 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     4501 cagcatccat gttggaattt aatcgcggcc tggagcaaga cgtttcccgt tgaatatggc
     4561 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgatgata
     4621 tatttttatc ttgtgcaatg taacatcaga gattttgaga cacaacgtgg ctttgttgaa
     4681 taaatcgaac ttttgctgag ttgaaggatc agtcatgacc aaaatccctt aacgtgagtt
     4741 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4801 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4861 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4921 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4981 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     5041 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     5101 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     5161 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     5221 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     5281 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     5341 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     5401 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     5461 ttctgtggat aaccgtgcgg ccgcccct
//
