LOCUS       sequence-309839-        4630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low-copy expression vector for K. marxianus, uracil selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4630)
  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 4630)
  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-URA
FEATURES             Location/Qualifiers
     source          1..4630
                     /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"
     CDS             1579..2382
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     CDS             complement(2895..3710)
                     /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(2895..3710)
                     /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     3618..3637
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3702..3721
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3935..4523
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4424..4443
                     /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 acggtttcct tgaaattttt ttgattcggt aatctccgaa
     1441 cagaaggaag aacgaaggaa ggagcacaga cttagattgg tatatatacg catatgtagt
     1501 gttgaagaaa catgaaattg cccagtattc ttaacccaac tgcacagaac aaaaacgtgc
     1561 aggaaacgaa gataaatcat gtcgaaagct acatataagg aacgtgctgc tactcatcct
     1621 agtcctgttg ctgccaagct atttaatatc atgcacgaaa agcaaacaaa cttgtgtgct
     1681 tcattggatg ttcgtaccac caaggaatta ctggagttag ttgaagcatt aggtcccaaa
     1741 atttgtttac taaaaacaca tgtggatatc ttgactgatt tttccatgga gggcacagtt
     1801 aagccgctaa aggcattatc cgccaagtac aattttttac tcttcgagga cagaaaattt
     1861 gctgacattg gtaatacagt caaattgcag tactctgcgg gtgtatacag aatagcagaa
     1921 tgggcagaca ttacgaatgc acacggtgtg gtgggcccag gtattgttag cggtttgaag
     1981 caggcggcag aagaagtaac aaaggaacct agaggccttt tgatgttagc agaattgtca
     2041 tgcaagggct ccctatctac tggagaatat actaagggta ctgttgacat tgcgaagagc
     2101 gacaaagatt ttgttatcgg ctttattgct caaagagaca tgggtggaag agatgaaggt
     2161 tacgattggt tgattatgac acccggtgtg ggtttagatg acaagggaga tgcattgggt
     2221 caacagtata gaaccgtgga tgatgtggtt tctacaggat ctgacattat tattgttgga
     2281 agaggactat ttgcaaaggg aagggatgct aaggtagagg gtgaacgtta cagaaaagca
     2341 ggctgggaag catatttgag aagatgcggc cagcaaaact aaaaaactgt attataagta
     2401 aatgcatgta tactaaactc acaaattaga gcttcaattt aattatatca gttattaccc
     2461 gagtttctac gcaagacttc ttgaagtgaa aaccaacttt cagtcttcaa actaaaaatg
     2521 aaaatcagtg ggctaaattc agcataatac ctgtggatat aattacatac aaaaatataa
     2581 ttaaaaatat acgttaaaaa taatttattt ttgtaaaaac tataaaatat attttacttt
     2641 cggaacaact ttttaactta taattttgtt ttaaataaaa acattgtatt taaaaataat
     2701 aaattattaa ataaaatttt aaagtatgtt tatttaaaat atagaatact attaaaatta
     2761 ctttaaactt taaaataaaa aaataataaa ttatatagtt ttaaaattaa taaattttat
     2821 acacgtgacc agatcataat agttttcttt ccttgccgag cggccgcgtg ttacaaccaa
     2881 ttaaccaatt ctgattagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     2941 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     3001 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     3061 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     3121 ccatgagtga cgactgaatc cggtgagaat ggcaaaagct tatgcatttc tttccagact
     3181 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     3241 ttcattcgtg attgcgcctg agcgaggcga aatacgcgat cgctgttaaa aggacaatta
     3301 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     3361 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     3421 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     3481 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     3541 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     3601 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     3661 gaatttaatc gcggcctgga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     3721 gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt tttatcttgt
     3781 gcaatgtaac atcagagatt ttgagacaca acgtggcttt gttgaataaa tcgaactttt
     3841 gctgagttga aggatcagtc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3901 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3961 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4021 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4081 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4141 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4201 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4261 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4321 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4381 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4441 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4501 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4561 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4621 gtgcggccgc
//
