LOCUS       sequence-317736-        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             343..354
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             361..1074
                     /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      1086..1157
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1173..1200
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             1589..2392
                     /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(2905..3720)
                     /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(2905..3720)
                     /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     3628..3647
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3712..3731
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3945..4533
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4434..4453
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtgcggccgc ccctgaattc gcatctagac tgatgagacg tggtagagcc acaaacagcc
       61 ggtacaagca acgatctcca ggaccatctg aatcatgcgc ggatgacacg aactcacgac
      121 ggcgatcaca gacattaacc cacagtacag acactgcgac aacgtggcaa ttcgtcgcaa
      181 tacaacgtga gaccgaaagt gaaacgtgat ttcatgcgtc attttgaaca ttttgtaaat
      241 cttatttaat aatgtgtgcg gcaattcaca tttaatttat gaatgttttc ttaacatcgc
      301 ggcaactcaa gaaacggcag gttcggatct tagctactag agaaagagga gaaatactag
      361 atgcgtaaag gcgaagagct gttcactggt gtcgtcccta ttctggtgga actggatggt
      421 gatgtcaacg gtcataagtt ttccgtgcgt ggcgagggtg aaggtgacgc aactaatggt
      481 aaactgacgc tgaagttcat ctgtactact ggtaaactgc cggttccttg gccgactctg
      541 gtaacgacgc tgacttatgg tgttcagtgc tttgctcgtt atccggacca tatgaagcag
      601 catgacttct tcaagtccgc catgccggaa ggctatgtgc aggaacgcac gatttccttt
      661 aaggatgacg gcacgtacaa aacgcgtgcg gaagtgaaat ttgaaggcga taccctggta
      721 aaccgcattg agctgaaagg cattgacttt aaagaggacg gcaatatcct gggccataag
      781 ctggaataca attttaacag ccacaatgtt tacatcaccg ccgataaaca aaaaaatggc
      841 attaaagcga attttaaaat tcgccacaac gtggaggatg gcagcgtgca gctggctgat
      901 cactaccagc aaaacactcc aatcggtgat ggtcctgttc tgctgccaga caatcactat
      961 ctgagcacgc aaagcgttct gtctaaagat ccgaacgaga aacgcgatca tatggttctg
     1021 ctggagttcg taaccgcagc gggcatcacg catggtatgg atgaactgta caaatgacca
     1081 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     1141 tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg
     1201 tttataggtc tcagctggaa atctgctcgt cagtggtgct cacactgacg aatcatgtac
     1261 agatcatacc gatgactgcc tggcgactca caactaagca agacagccgg aaccagcgcc
     1321 ggcgaacacc actgcatata tggcatatca caacagtcca cgtctcaagc agttacagag
     1381 atgttacgaa ccactagtgc actgcagtac acggtttcct tgaaattttt ttgattcggt
     1441 aatctccgaa cagaaggaag aacgaaggaa ggagcacaga cttagattgg tatatatacg
     1501 catatgtagt gttgaagaaa catgaaattg cccagtattc ttaacccaac tgcacagaac
     1561 aaaaacgtgc aggaaacgaa gataaatcat gtcgaaagct acatataagg aacgtgctgc
     1621 tactcatcct agtcctgttg ctgccaagct atttaatatc atgcacgaaa agcaaacaaa
     1681 cttgtgtgct tcattggatg ttcgtaccac caaggaatta ctggagttag ttgaagcatt
     1741 aggtcccaaa atttgtttac taaaaacaca tgtggatatc ttgactgatt tttccatgga
     1801 gggcacagtt aagccgctaa aggcattatc cgccaagtac aattttttac tcttcgagga
     1861 cagaaaattt gctgacattg gtaatacagt caaattgcag tactctgcgg gtgtatacag
     1921 aatagcagaa tgggcagaca ttacgaatgc acacggtgtg gtgggcccag gtattgttag
     1981 cggtttgaag caggcggcag aagaagtaac aaaggaacct agaggccttt tgatgttagc
     2041 agaattgtca tgcaagggct ccctatctac tggagaatat actaagggta ctgttgacat
     2101 tgcgaagagc gacaaagatt ttgttatcgg ctttattgct caaagagaca tgggtggaag
     2161 agatgaaggt tacgattggt tgattatgac acccggtgtg ggtttagatg acaagggaga
     2221 tgcattgggt caacagtata gaaccgtgga tgatgtggtt tctacaggat ctgacattat
     2281 tattgttgga agaggactat ttgcaaaggg aagggatgct aaggtagagg gtgaacgtta
     2341 cagaaaagca ggctgggaag catatttgag aagatgcggc cagcaaaact aaaaaactgt
     2401 attataagta aatgcatgta tactaaactc acaaattaga gcttcaattt aattatatca
     2461 gttattaccc gagtttctac gcaagacttc ttgaagtgaa aaccaacttt cagtcttcaa
     2521 actaaaaatg aaaatcagtg ggctaaattc agcataatac ctgtggatat aattacatac
     2581 aaaaatataa ttaaaaatat acgttaaaaa taatttattt ttgtaaaaac tataaaatat
     2641 attttacttt cggaacaact ttttaactta taattttgtt ttaaataaaa acattgtatt
     2701 taaaaataat aaattattaa ataaaatttt aaagtatgtt tatttaaaat atagaatact
     2761 attaaaatta ctttaaactt taaaataaaa aaataataaa ttatatagtt ttaaaattaa
     2821 taaattttat acacgtgacc agatcataat agttttcttt ccttgccgag cggccgcgtg
     2881 ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg agcatcaaat gaaactgcaa
     2941 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     3001 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     3061 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     3121 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagct tatgcatttc
     3181 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     3241 caaaccgtta ttcattcgtg attgcgcctg agcgaggcga aatacgcgat cgctgttaaa
     3301 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     3361 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     3421 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     3481 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     3541 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     3601 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     3661 atccatgttg gaatttaatc gcggcctgga gcaagacgtt tcccgttgaa tatggctcat
     3721 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt
     3781 tttatcttgt gcaatgtaac atcagagatt ttgagacaca acgtggcttt gttgaataaa
     3841 tcgaactttt gctgagttga aggatcagtc atgaccaaaa tcccttaacg tgagttttcg
     3901 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3961 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4021 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4081 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4141 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4201 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4261 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4321 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4381 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4441 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4501 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4561 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4621 gtggataacc
//
