LOCUS       sequence-317072-        5185 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low-copy expression vector for K. marxianus, hygromycin selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5185)
  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 5185)
  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-HPH
FEATURES             Location/Qualifiers
     source          1..5185
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             357..368
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             375..1088
                     /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      1100..1171
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1187..1214
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     gene            1465..3039
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     CDS             1809..2837
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1809..2837
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      2842..3039
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(3474..4289)
                     /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(3474..4289)
                     /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     4197..4216
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4281..4300
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4514..5102
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5003..5022
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctgtggat aaccgtgcgg ccgcccctga attcgcatct agactgatga gacgtggtag
       61 agccacaaac agccggtaca agcaacgatc tccaggacca tctgaatcat gcgcggatga
      121 cacgaactca cgacggcgat cacagacatt aacccacagt acagacactg cgacaacgtg
      181 gcaattcgtc gcaatacaac gtgagaccga aagtgaaacg tgatttcatg cgtcattttg
      241 aacattttgt aaatcttatt taataatgtg tgcggcaatt cacatttaat ttatgaatgt
      301 tttcttaaca tcgcggcaac tcaagaaacg gcaggttcgg atcttagcta ctagagaaag
      361 aggagaaata ctagatgcgt aaaggcgaag agctgttcac tggtgtcgtc cctattctgg
      421 tggaactgga tggtgatgtc aacggtcata agttttccgt gcgtggcgag ggtgaaggtg
      481 acgcaactaa tggtaaactg acgctgaagt tcatctgtac tactggtaaa ctgccggttc
      541 cttggccgac tctggtaacg acgctgactt atggtgttca gtgctttgct cgttatccgg
      601 accatatgaa gcagcatgac ttcttcaagt ccgccatgcc ggaaggctat gtgcaggaac
      661 gcacgatttc ctttaaggat gacggcacgt acaaaacgcg tgcggaagtg aaatttgaag
      721 gcgataccct ggtaaaccgc attgagctga aaggcattga ctttaaagag gacggcaata
      781 tcctgggcca taagctggaa tacaatttta acagccacaa tgtttacatc accgccgata
      841 aacaaaaaaa tggcattaaa gcgaatttta aaattcgcca caacgtggag gatggcagcg
      901 tgcagctggc tgatcactac cagcaaaaca ctccaatcgg tgatggtcct gttctgctgc
      961 cagacaatca ctatctgagc acgcaaagcg ttctgtctaa agatccgaac gagaaacgcg
     1021 atcatatggt tctgctggag ttcgtaaccg cagcgggcat cacgcatggt atggatgaac
     1081 tgtacaaatg accaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     1141 ttttatctgt tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg
     1201 tgggcctttc tgcgtttata ggtctcagct ggaaatctgc tcgtcagtgg tgctcacact
     1261 gacgaatcat gtacagatca taccgatgac tgcctggcga ctcacaacta agcaagacag
     1321 ccggaaccag cgccggcgaa caccactgca tatatggcat atcacaacag tccacgtctc
     1381 aagcagttac agagatgtta cgaaccacta gtgcactgca gtacagttta gcttgcctcg
     1441 tccccgccgg gtcacccggc cagcgacatg gaggcccaga ataccctcct tgacagtctt
     1501 gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt acatttagcc catacatccc
     1561 catgtataat catttgcatc catacatttt gatggccgca cggcgcgaag caaaaattac
     1621 ggctcctcgc tccagacctg cgagcaggga aacgctcccc tcacagacgc gttgaattgt
     1681 ccccacgccg cgcccctgta gagaaatata aaaggttagg atttgccact gaggttcttc
     1741 tttcatatac ttccttttaa aatcttgcta ggatacagtt ctcacatcac atccgaacat
     1801 aaacaaaaat gggtaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     1861 aaaagttcga cagcgtgtcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     1921 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     1981 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     2041 tgcttgacat tggggaattt agcgagagcc tgacctattg catctcccgc cgtgcacagg
     2101 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcaa ccggtcgcgg
     2161 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     2221 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     2281 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     2341 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     2401 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     2461 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     2521 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     2581 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     2641 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     2701 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     2761 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     2821 cgagggcaaa ggaataaagt aactgacaat aaaaagattc ttgttttcaa gaacttgtca
     2881 tttgtatagt ttttttatat tgtagttgtt ctattttaat caaatgttag cgtgatttat
     2941 attttttttc gcctcgacat catctgccca gatgcgaagt taagtgcgca gaaagtaata
     3001 tcatgcgtca atcgtatgtg aatgctggtc gctatactgg agtttctacg caagacttct
     3061 tgaagtgaaa accaactttc agtcttcaaa ctaaaaatga aaatcagtgg gctaaattca
     3121 gcataatacc tgtggatata attacataca aaaatataat taaaaatata cgttaaaaat
     3181 aatttatttt tgtaaaaact ataaaatata ttttactttc ggaacaactt tttaacttat
     3241 aattttgttt taaataaaaa cattgtattt aaaaataata aattattaaa taaaatttta
     3301 aagtatgttt atttaaaata tagaatacta ttaaaattac tttaaacttt aaaataaaaa
     3361 aataataaat tatatagttt taaaattaat aaattttata cacgtgacca gatcataata
     3421 gttttctttc cttgccgagc ggccgcgtgt tacaaccaat taaccaattc tgattagaaa
     3481 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     3541 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     3601 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     3661 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     3721 ggtgagaatg gcaaaagctt atgcatttct ttccagactt gttcaacagg ccagccatta
     3781 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     3841 gcgaggcgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     3901 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     3961 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     4021 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     4081 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     4141 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     4201 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctggag
     4261 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     4321 gacagtttta ttgttcatga tgatatattt ttatcttgtg caatgtaaca tcagagattt
     4381 tgagacacaa cgtggctttg ttgaataaat cgaacttttg ctgagttgaa ggatcagtca
     4441 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4501 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4561 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4621 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     4681 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4741 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4801 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4861 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4921 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4981 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5041 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5101 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5161 tgttctttcc tgcgttatcc cctga
//
