LOCUS       sequence-318010-        4522 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High-copy expression vector for K. marxianus, histidine selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4522)
  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 4522)
  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-HIS-C4
FEATURES             Location/Qualifiers
     source          1..4522
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      611..682
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      698..725
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        986..1173
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             1174..1836
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     CDS             complement(2322..3137)
                     /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(2322..3137)
                     /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     3045..3064
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3129..3148
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3362..3950
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3851..3870
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             4390..4401
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(4408..4522,1..599)
                     /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 atggtaaact gacgctgaag ttcatctgta ctactggtaa actgccggtt ccttggccga
       61 ctctggtaac gacgctgact tatggtgttc agtgctttgc tcgttatccg gaccatatga
      121 agcagcatga cttcttcaag tccgccatgc cggaaggcta tgtgcaggaa cgcacgattt
      181 cctttaagga tgacggcacg tacaaaacgc gtgcggaagt gaaatttgaa ggcgataccc
      241 tggtaaaccg cattgagctg aaaggcattg actttaaaga ggacggcaat atcctgggcc
      301 ataagctgga atacaatttt aacagccaca atgtttacat caccgccgat aaacaaaaaa
      361 atggcattaa agcgaatttt aaaattcgcc acaacgtgga ggatggcagc gtgcagctgg
      421 ctgatcacta ccagcaaaac actccaatcg gtgatggtcc tgttctgctg ccagacaatc
      481 actatctgag cacgcaaagc gttctgtcta aagatccgaa cgagaaacgc gatcatatgg
      541 ttctgctgga gttcgtaacc gcagcgggca tcacgcatgg tatggatgaa ctgtacaaat
      601 gaccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
      661 ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt
      721 ctgcgtttat aggtctcagc tggaaatctg ctcgtcagtg gtgctcacac tgacgaatca
      781 tgtacagatc ataccgatga ctgcctggcg actcacaact aagcaagaca gccggaacca
      841 gcgccggcga acaccactgc atatatggca tatcacaaca gtccacgtct caagcagtta
      901 cagagatgtt acgaaccact agtgcactgc agtacaaaca cagtcctttc ccgcaatttt
      961 ctttttctat tactcttggc ctcctctagt acactctata tttttttatg cctcggtaat
     1021 gattttcatt tttttttttc cacctagcgg atgactcttt ttttttctta gcgattggca
     1081 ttatcacata atgaattata cattatataa agtaatgtga tttcttcgaa gaatatacta
     1141 aaaaatgagc aggcaagata aacgaaggca aagatgacag agcagaaagc cctagtaaag
     1201 cgtattacaa atgaaaccaa gattcagatt gcgatctctt taaagggtgg tcccctagcg
     1261 atagagcact cgatcttccc agaaaaagag gcagaagcag tagcagaaca ggccacacaa
     1321 tcgcaagtga ttaacgtcca cacaggtata gggtttctgg accatatgat acatgctctg
     1381 gccaagcatt ccggctggtc gctaatcgtt gagtgcattg gtgacttaca catagacgac
     1441 catcacacca ctgaggactg cgggattgct ctcggtcaag cttttaaaga ggccctaggg
     1501 gccgtgcgtg gagtaaaaag gtttggatca ggatttgcgc ctttggatga ggcactttcc
     1561 agagcggtgg ttgatctttc gaacaggccg tacgcagttg tcgaacttgg tttgcaaagg
     1621 gagaaagtag gtgatctctc ttgcgagatg atcccgcatt ttcttgaaag ctttgcagag
     1681 gctagcagaa ttaccctcca cgttgattgt ctgcgaggca agaatgatca tcaccgtagt
     1741 gagagtgcgt tcaaggctct tgcggttgcc ataagagaag ccacctcgcc caatggtacc
     1801 aacgatgttc cctccaccaa aggtgttctt atgtagtgac accgattatt taaagctgct
     1861 gcatacgata tatatacatg tgtatatatg tatacctatg aatgtcagta agtatgtata
     1921 cgaacagtat gatactgaag atgacaaggt aatgcatcat tctatacgtg tcattctgaa
     1981 cgaggcgcgc tttccttttt tctttttgct ttttcttttt ttttctcttg aactcgacgg
     2041 atcatagagt caatactttc taacattgac cattttgcat cattttgtta cggaaaagtg
     2101 ctttccaatc atatataatt aaaactagcg gatcacatta aaaagttcat ccaaagtgaa
     2161 aaaacattct aaatttagta gacagcaatt gttttaaaag ggtgttccta ttttcatata
     2221 acggagagag ttaagcttcc tgtatgcaat tagcaaatat tttcactgta gagttctggg
     2281 acccgagcgg ccgcgtgtta caaccaatta accaattctg attagaaaaa ctcatcgagc
     2341 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     2401 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     2461 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     2521 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     2581 aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     2641 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gaggcgaaat
     2701 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     2761 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     2821 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     2881 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     2941 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     3001 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     3061 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctggagca agacgtttcc
     3121 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     3181 gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg
     3241 tggctttgtt gaataaatcg aacttttgct gagttgaagg atcagtcatg accaaaatcc
     3301 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3361 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3421 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3481 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     3541 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3601 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3661 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3721 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3781 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3841 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     3901 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3961 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4021 cgttatcccc tgattctgtg gataaccgtg cggccgcccc tgaattcgca tctagactga
     4081 tgagacgtgg tagagccaca aacagccggt acaagcaacg atctccagga ccatctgaat
     4141 catgcgcgga tgacacgaac tcacgacggc gatcacagac attaacccac agtacagaca
     4201 ctgcgacaac gtggcaattc gtcgcaatac aacgtgagac cgaaagtgaa acgtgatttc
     4261 atgcgtcatt ttgaacattt tgtaaatctt atttaataat gtgtgcggca attcacattt
     4321 aatttatgaa tgttttctta acatcgcggc aactcaagaa acggcaggtt cggatcttag
     4381 ctactagaga aagaggagaa atactagatg cgtaaaggcg aagagctgtt cactggtgtc
     4441 gtccctattc tggtggaact ggatggtgat gtcaacggtc ataagttttc cgtgcgtggc
     4501 gagggtgaag gtgacgcaac ta
//
