LOCUS       sequence-317968-        4471 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High-copy expression vector for K. marxianus, uracil selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4471)
  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 4471)
  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-C4
FEATURES             Location/Qualifiers
     source          1..4471
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      656..727
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      743..770
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             1159..1962
                     /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(2316..3131)
                     /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(2316..3131)
                     /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     3039..3058
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3123..3142
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3356..3944
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3845..3864
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             4384..4395
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(4402..4471,1..644)
                     /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 gtcataagtt ttccgtgcgt ggcgagggtg aaggtgacgc aactaatggt aaactgacgc
       61 tgaagttcat ctgtactact ggtaaactgc cggttccttg gccgactctg gtaacgacgc
      121 tgacttatgg tgttcagtgc tttgctcgtt atccggacca tatgaagcag catgacttct
      181 tcaagtccgc catgccggaa ggctatgtgc aggaacgcac gatttccttt aaggatgacg
      241 gcacgtacaa aacgcgtgcg gaagtgaaat ttgaaggcga taccctggta aaccgcattg
      301 agctgaaagg cattgacttt aaagaggacg gcaatatcct gggccataag ctggaataca
      361 attttaacag ccacaatgtt tacatcaccg ccgataaaca aaaaaatggc attaaagcga
      421 attttaaaat tcgccacaac gtggaggatg gcagcgtgca gctggctgat cactaccagc
      481 aaaacactcc aatcggtgat ggtcctgttc tgctgccaga caatcactat ctgagcacgc
      541 aaagcgttct gtctaaagat ccgaacgaga aacgcgatca tatggttctg ctggagttcg
      601 taaccgcagc gggcatcacg catggtatgg atgaactgta caaatgacca ggcatcaaat
      661 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
      721 cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg tttataggtc
      781 tcagctggaa atctgctcgt cagtggtgct cacactgacg aatcatgtac agatcatacc
      841 gatgactgcc tggcgactca caactaagca agacagccgg aaccagcgcc ggcgaacacc
      901 actgcatata tggcatatca caacagtcca cgtctcaagc agttacagag atgttacgaa
      961 ccactagtgc actgcagtac acggtttcct tgaaattttt ttgattcggt aatctccgaa
     1021 cagaaggaag aacgaaggaa ggagcacaga cttagattgg tatatatacg catatgtagt
     1081 gttgaagaaa catgaaattg cccagtattc ttaacccaac tgcacagaac aaaaacgtgc
     1141 aggaaacgaa gataaatcat gtcgaaagct acatataagg aacgtgctgc tactcatcct
     1201 agtcctgttg ctgccaagct atttaatatc atgcacgaaa agcaaacaaa cttgtgtgct
     1261 tcattggatg ttcgtaccac caaggaatta ctggagttag ttgaagcatt aggtcccaaa
     1321 atttgtttac taaaaacaca tgtggatatc ttgactgatt tttccatgga gggcacagtt
     1381 aagccgctaa aggcattatc cgccaagtac aattttttac tcttcgagga cagaaaattt
     1441 gctgacattg gtaatacagt caaattgcag tactctgcgg gtgtatacag aatagcagaa
     1501 tgggcagaca ttacgaatgc acacggtgtg gtgggcccag gtattgttag cggtttgaag
     1561 caggcggcag aagaagtaac aaaggaacct agaggccttt tgatgttagc agaattgtca
     1621 tgcaagggct ccctatctac tggagaatat actaagggta ctgttgacat tgcgaagagc
     1681 gacaaagatt ttgttatcgg ctttattgct caaagagaca tgggtggaag agatgaaggt
     1741 tacgattggt tgattatgac acccggtgtg ggtttagatg acaagggaga tgcattgggt
     1801 caacagtata gaaccgtgga tgatgtggtt tctacaggat ctgacattat tattgttgga
     1861 agaggactat ttgcaaaggg aagggatgct aaggtagagg gtgaacgtta cagaaaagca
     1921 ggctgggaag catatttgag aagatgcggc cagcaaaact aaaaaactgt attataagta
     1981 aatgcatgta tactaaactc acaaattaga gcttcaattt aattatatca gttattaccc
     2041 gagtcaatac tttctaacat tgaccatttt gcatcatttt gttacggaaa agtgctttcc
     2101 aatcatatat aattaaaact agcggatcac attaaaaagt tcatccaaag tgaaaaaaca
     2161 ttctaaattt agtagacagc aattgtttta aaagggtgtt cctattttca tataacggag
     2221 agagttaagc ttcctgtatg caattagcaa atattttcac tgtagagttc tgggacccga
     2281 gcggccgcgt gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa
     2341 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     2401 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     2461 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     2521 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc
     2581 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     2641 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgaggcg aaatacgcga
     2701 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     2761 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     2821 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     2881 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     2941 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     3001 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     3061 tataaatcag catccatgtt ggaatttaat cgcggcctgg agcaagacgt ttcccgttga
     3121 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     3181 gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt
     3241 tgttgaataa atcgaacttt tgctgagttg aaggatcagt catgaccaaa atcccttaac
     3301 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3361 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3421 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3481 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     3541 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3601 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3661 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3721 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3781 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3841 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3901 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3961 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4021 cccctgattc tgtggataac cgtgcggccg cccctgaatt cgcatctaga ctgatgagac
     4081 gtggtagagc cacaaacagc cggtacaagc aacgatctcc aggaccatct gaatcatgcg
     4141 cggatgacac gaactcacga cggcgatcac agacattaac ccacagtaca gacactgcga
     4201 caacgtggca attcgtcgca atacaacgtg agaccgaaag tgaaacgtga tttcatgcgt
     4261 cattttgaac attttgtaaa tcttatttaa taatgtgtgc ggcaattcac atttaattta
     4321 tgaatgtttt cttaacatcg cggcaactca agaaacggca ggttcggatc ttagctacta
     4381 gagaaagagg agaaatacta gatgcgtaaa ggcgaagagc tgttcactgg tgtcgtccct
     4441 attctggtgg aactggatgg tgatgtcaac g
//
