LOCUS       sequence-326325-        6114 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting integration site I2 
            described in (Rajkumar et al.,2019). Uracil selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6114)
  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 6114)
  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: pI2-MTU-DO-URA
FEATURES             Location/Qualifiers
     source          1..6114
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1077..1892)
                     /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(1077..1892)
                     /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     1800..1819
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1884..1903
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2117..2705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2606..2625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             4013..4024
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4031..4744
                     /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      4756..4827
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4843..4870
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             5259..6062
                     /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"
ORIGIN
        1 caatttaatt atatcagtta ttacccgagt tgaagcatag aagaaatgtt taaccaaaaa
       61 ctatcctttt tatttatttt gaattttctc tctcatattt ttgtaaaacg atactttaac
      121 cacactcaag cttttgcttg ctactcaaat acctgaaatc tttatagaca attttccttc
      181 ttattcgtaa taataaaact atattgttct cttcctgttt ttttttttaa aaaaaaaaac
      241 tgtcaaaatc tcccccacag aaaaataaaa acaaattata cttttgggag taaaccatga
      301 acttaaaaca acagttcata ttcttaagta gtgccactat agttaaccag agacaattta
      361 actcctaaat cttcatactg aagattgaca taaactaaat aatgactaca ctgttacttt
      421 tgagtcagtt gggtcttttg gcattcgcca atgctattaa ccctatggta aagggcgaat
      481 atagaattga acatcctaag ataggagact tgtcagaaat tgaatctgct atctctccca
      541 aatctttcta tgaatcattt caatatggtt ccgaagcgga gttccgtaag aattgggttt
      601 cttctgatct aacaatggaa catgtagacg aaacatcttc gaaggcgttc cctggcacat
      661 ggaagttgca gtcaccattt ttggtcccag gattcagtga tgatctatct ttagttttgg
      721 gtagcgaaca aactagagct gctattgccc ataggttaaa atacgatcta tcgatcaagg
      781 ataatgagaa actagtggtg caatatgaag ttaaacttca aaaaatgctt gaatgtggcg
      841 gtgcatatat caaattgttg aaaacctccg caactgatct ttctcagtac gaccactcgt
      901 cagatgacta tgctgtcatc tttggcccag acttttgcca aggttatgtg gacgaaattc
      961 atttggtttt gaagcgcgag aatccttttt ctggaaaatc tgaagacaaa ttcttattaa
     1021 atgccggcgc gcctcagccg agcggccgcg tgttacaacc aattaaccaa ttctgattag
     1081 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     1141 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     1201 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     1261 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     1321 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca
     1381 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     1441 tgagcgaggc gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     1501 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     1561 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     1621 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     1681 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     1741 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     1801 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctg
     1861 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     1921 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga
     1981 ttttgagaca caacgtggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag
     2041 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2101 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2161 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2221 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     2281 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     2341 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     2401 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     2461 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     2521 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     2581 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     2641 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     2701 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     2761 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtgcggcc gcccctttca
     2821 ggcgcgccac cgaatattaa ataatctttt tttatttctt atgtacgcct gctttgtatg
     2881 atacaaaatc atcgtatcgt attcggcttc atgttatccg tcgaatttag attaccttgg
     2941 ctaaggggcc aaggtggatg attcgtatga gtatcacaat ttacaaatta caagttttgc
     3001 aaagagtatg taaactgtaa aaaagaaaac attgacaaat atacctgaaa agagtccatt
     3061 ttctacattt gagaaaaaaa ggaataaagt gattctcctc atcgctcata aaaaaatact
     3121 attcatgagc aaaattgaga aaaatggcaa tcacattcaa tactagttag atagccatat
     3181 ctaaactgct gtacgtggtt taatactagc accaacatca tttgaataat ggcgaagaaa
     3241 ccaatttcga aacattcccg tgcggcaaga agatcagaag cagccgaacc ggaggctaag
     3301 gctttagaga ctttacccag agctgaaaag acagattttg cagcaacatt aatcagaact
     3361 gctaacaaaa acgaagcttt gctcgaagcg aagatccaaa agcgtggaca aaaaaataag
     3421 aagaaaaacg cgggaagagt gagtaagcaa cttatagatg acagttcttt aacaagtagg
     3481 aaagctattc gtgccttaaa tattaccagt aggctcgatg gtaagaggga gaaagccatt
     3541 catagagcta aatatgtgca gtcagctcgt aaagctggat gggatactac aaaccaaagc
     3601 attaggaaag aattatcgac actacaaaac gcagcaaata cggtcagcga ttccgttaac
     3661 aaaagcacgg acaaggaagg gtctgcgaat actgaagatg cgatgataga agacgatcaa
     3721 gatcacattg aaatttacga cagtgctaaa agccagaagg tacaagaaaa agcagtatca
     3781 accactttta cggcatcaaa cgtatttacg tcattagagg atgagatgaa ttcgcatcta
     3841 gactgatgag acgaacgtga gaccgaaagt gaaacgtgat ttcatgcgtc attttgaaca
     3901 ttttgtaaat cttatttaat aatgtgtgcg gcaattcaca tttaatttat gaatgttttc
     3961 ttaacatcgc ggcaactcaa gaaacggcag gttcggatct tagctactag agaaagagga
     4021 gaaatactag atgcgtaaag gcgaagagct gttcactggt gtcgtcccta ttctggtgga
     4081 actggatggt gatgtcaacg gtcataagtt ttccgtgcgt ggcgagggtg aaggtgacgc
     4141 aactaatggt aaactgacgc tgaagttcat ctgtactact ggtaaactgc cggttccttg
     4201 gccgactctg gtaacgacgc tgacttatgg tgttcagtgc tttgctcgtt atccggacca
     4261 tatgaagcag catgacttct tcaagtccgc catgccggaa ggctatgtgc aggaacgcac
     4321 gatttccttt aaggatgacg gcacgtacaa aacgcgtgcg gaagtgaaat ttgaaggcga
     4381 taccctggta aaccgcattg agctgaaagg cattgacttt aaagaggacg gcaatatcct
     4441 gggccataag ctggaataca attttaacag ccacaatgtt tacatcaccg ccgataaaca
     4501 aaaaaatggc attaaagcga attttaaaat tcgccacaac gtggaggatg gcagcgtgca
     4561 gctggctgat cactaccagc aaaacactcc aatcggtgat ggtcctgttc tgctgccaga
     4621 caatcactat ctgagcacgc aaagcgttct gtctaaagat ccgaacgaga aacgcgatca
     4681 tatggttctg ctggagttcg taaccgcagc gggcatcacg catggtatgg atgaactgta
     4741 caaatgacca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     4801 atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg
     4861 cctttctgcg tttataggtc tcagctggaa atctgctcgt cagtggtgct cacactgacg
     4921 aatcatgtac agatcatacc gatgactgcc tggcgactca caactaagca agacagccgg
     4981 aaccagcgcc ggcgaacacc actgcatata tggcatatca caacagtcca cgtctcaagc
     5041 agttacagag atgttacgaa ccactagtgc actgcagtac acggtttcct tgaaattttt
     5101 ttgattcggt aatctccgaa cagaaggaag aacgaaggaa ggagcacaga cttagattgg
     5161 tatatatacg catatgtagt gttgaagaaa catgaaattg cccagtattc ttaacccaac
     5221 tgcacagaac aaaaacgtgc aggaaacgaa gataaatcat gtcgaaagct acatataagg
     5281 aacgtgctgc tactcatcct agtcctgttg ctgccaagct atttaatatc atgcacgaaa
     5341 agcaaacaaa cttgtgtgct tcattggatg ttcgtaccac caaggaatta ctggagttag
     5401 ttgaagcatt aggtcccaaa atttgtttac taaaaacaca tgtggatatc ttgactgatt
     5461 tttccatgga gggcacagtt aagccgctaa aggcattatc cgccaagtac aattttttac
     5521 tcttcgagga cagaaaattt gctgacattg gtaatacagt caaattgcag tactctgcgg
     5581 gtgtatacag aatagcagaa tgggcagaca ttacgaatgc acacggtgtg gtgggcccag
     5641 gtattgttag cggtttgaag caggcggcag aagaagtaac aaaggaacct agaggccttt
     5701 tgatgttagc agaattgtca tgcaagggct ccctatctac tggagaatat actaagggta
     5761 ctgttgacat tgcgaagagc gacaaagatt ttgttatcgg ctttattgct caaagagaca
     5821 tgggtggaag agatgaaggt tacgattggt tgattatgac acccggtgtg ggtttagatg
     5881 acaagggaga tgcattgggt caacagtata gaaccgtgga tgatgtggtt tctacaggat
     5941 ctgacattat tattgttgga agaggactat ttgcaaaggg aagggatgct aaggtagagg
     6001 gtgaacgtta cagaaaagca ggctgggaag catatttgag aagatgcggc cagcaaaact
     6061 aaaaaactgt attataagta aatgcatgta tactaaactc acaaattaga gctt
//
