LOCUS       sequence-310500-        4471 bp    DNA     circular SYN 14-APR-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 Apr 14, 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"
     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(2736..3551)
                     /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(2736..3551)
                     /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     3459..3478
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3543..3562
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3776..4364
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4265..4284
                     /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 gagtcaatac tttctaacat tgaccatttt gcatcatttt gttacggaaa agtgctttcc
     2521 aatcatatat aattaaaact agcggatcac attaaaaagt tcatccaaag tgaaaaaaca
     2581 ttctaaattt agtagacagc aattgtttta aaagggtgtt cctattttca tataacggag
     2641 agagttaagc ttcctgtata caattagcaa atattttcac tatagagacc tgggacccga
     2701 gcggccgcgt gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa
     2761 tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc
     2821 tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg
     2881 tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata
     2941 aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc
     3001 ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca
     3061 ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgaggcg aaatacgcga
     3121 tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc
     3181 agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt
     3241 ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg
     3301 atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca
     3361 tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca
     3421 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
     3481 tataaatcag catccatgtt ggaatttaat cgcggcctgg agcaagacgt ttcccgttga
     3541 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
     3601 gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt
     3661 tgttgaataa atcgaacttt tgctgagttg aaggatcagt catgaccaaa atcccttaac
     3721 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3781 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3841 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3901 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     3961 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4021 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4081 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4141 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4201 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4261 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4321 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4381 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4441 cccctgattc tgtggataac cgtgcggccg c
//
