LOCUS       sequence-318012-        4807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High-copy expression vector for K. marxianus, G418 selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4807)
  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 4807)
  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-G418-C4
FEATURES             Location/Qualifiers
     source          1..4807
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      270..341
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      357..384
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     gene            635..1990
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     CDS             979..1788
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      1793..1990
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(2266..3081)
                     /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(2266..3081)
                     /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     3073..3092
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3306..3894
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3795..3814
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             4334..4345
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(4352..4807,1..258)
                     /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 accgccgata aacaaaaaaa tggcattaaa gcgaatttta aaattcgcca caacgtggag
       61 gatggcagcg tgcagctggc tgatcactac cagcaaaaca ctccaatcgg tgatggtcct
      121 gttctgctgc cagacaatca ctatctgagc acgcaaagcg ttctgtctaa agatccgaac
      181 gagaaacgcg atcatatggt tctgctggag ttcgtaaccg cagcgggcat cacgcatggt
      241 atggatgaac tgtacaaatg accaggcatc aaataaaacg aaaggctcag tcgaaagact
      301 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct ctactagagt cacactggct
      361 caccttcggg tgggcctttc tgcgtttata ggtctcagct ggaaatctgc tcgtcagtgg
      421 tgctcacact gacgaatcat gtacagatca taccgatgac tgcctggcga ctcacaacta
      481 agcaagacag ccggaaccag cgccggcgaa caccactgca tatatggcat atcacaacag
      541 tccacgtctc aagcagttac agagatgtta cgaaccacta gtgcactgca gtacagttta
      601 gcttgcctcg tccccgccgg gtcacccggc cagcgacatg gaggcccaga ataccctcct
      661 tgacagtctt gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt acatttagcc
      721 catacatccc catgtataat catttgcatc catacatttt gatggccgca cggcgcgaag
      781 caaaaattac ggctcctcgc tccagacctg cgagcaggga aacgctcccc tcacagacgc
      841 gttgaattgt ccccacgccg cgcccctgta gagaaatata aaaggttagg atttgccact
      901 gaggttcttc tttcatatac ttccttttaa aatcttgcta ggatacagtt ctcacatcac
      961 atccgaacat aaacaaaaat gggtaaggaa aagactcacg tttcgaggcc gcgattaaat
     1021 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
     1081 ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
     1141 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
     1201 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     1261 ctcaccactg cgatccccgg caaaacagca ttccaggtat tagaagaata tcctgattca
     1321 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
     1381 tgtaattgtc cttttaacag cgatcgcgta tttcgtctgg ctcaggcgca atcacgaatg
     1441 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     1501 caagtctgga aagaaatgca taagcttttg ccattctcac cggattcagt cgtcactcat
     1561 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
     1621 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
     1681 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
     1741 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaaag taactgacaa
     1801 taaaaagatt cttgttttca agaacttgtc atttgtatag tttttttata ttgtagttgt
     1861 tctattttaa tcaaatgtta gcgtgattta tatttttttt cgcctcgaca tcatctgccc
     1921 agatgcgaag ttaagtgcgc agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt
     1981 cgctatactg gagtcaatac tttctaacat tgaccatttt gcatcatttt gttacggaaa
     2041 agtgctttcc aatcatatat aattaaaact agcggatcac attaaaaagt tcatccaaag
     2101 tgaaaaaaca ttctaaattt agtagacagc aattgtttta aaagggtgtt cctattttca
     2161 tataacggag agagttaagc ttcctgtatg caattagcaa atattttcac tgtagagttc
     2221 tgggacccga gcggccgcgt gttacaacca attaaccaat tctgattaga aaaactcatc
     2281 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     2341 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     2401 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     2461 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     2521 tggcaaaagc ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     2581 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgaggcg
     2641 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     2701 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     2761 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     2821 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     2881 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     2941 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     3001 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctgg agcaagacgt
     3061 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     3121 tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac
     3181 aacgtggctt tgttgaataa atcgaacttt tgctgagttg aaggatcagt catgaccaaa
     3241 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3301 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3361 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3421 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     3481 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3541 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3601 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3661 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3721 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3781 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3841 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3901 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3961 cctgcgttat cccctgattc tgtggataac cgtgcggccg cccctgaatt cgcatctaga
     4021 ctgatgagac gtggtagagc cacaaacagc cggtacaagc aacgatctcc aggaccatct
     4081 gaatcatgcg cggatgacac gaactcacga cggcgatcac agacattaac ccacagtaca
     4141 gacactgcga caacgtggca attcgtcgca atacaacgtg agaccgaaag tgaaacgtga
     4201 tttcatgcgt cattttgaac attttgtaaa tcttatttaa taatgtgtgc ggcaattcac
     4261 atttaattta tgaatgtttt cttaacatcg cggcaactca agaaacggca ggttcggatc
     4321 ttagctacta gagaaagagg agaaatacta gatgcgtaaa ggcgaagagc tgttcactgg
     4381 tgtcgtccct attctggtgg aactggatgg tgatgtcaac ggtcataagt tttccgtgcg
     4441 tggcgagggt gaaggtgacg caactaatgg taaactgacg ctgaagttca tctgtactac
     4501 tggtaaactg ccggttcctt ggccgactct ggtaacgacg ctgacttatg gtgttcagtg
     4561 ctttgctcgt tatccggacc atatgaagca gcatgacttc ttcaagtccg ccatgccgga
     4621 aggctatgtg caggaacgca cgatttcctt taaggatgac ggcacgtaca aaacgcgtgc
     4681 ggaagtgaaa tttgaaggcg ataccctggt aaaccgcatt gagctgaaag gcattgactt
     4741 taaagaggac ggcaatatcc tgggccataa gctggaatac aattttaaca gccacaatgt
     4801 ttacatc
//
