LOCUS       sequence-318049-        6661 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting integration site I3 
            described in (Rajkumar et al.,2019). Leucine selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6661)
  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 6661)
  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: pI3-MTU-DO-LEU
FEATURES             Location/Qualifiers
     source          1..6661
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1056..1067
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1074..1787
                     /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      1799..1870
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1886..1913
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        2365..2772
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2773..3867
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     CDS             complement(4932..5747)
                     /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(4932..5747)
                     /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     5655..5674
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5739..5758
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5972..6560
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6461..6480
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggccgcccct ttcaggcgcg cctcataccg tttatgattg catttacgtt actgaccgga
       61 gttttgtccg tatttgtgaa gccgttggag ataattgagg caccaaggtt cgaaccagac
      121 tatgctatat tctacccagg aattaattgc cagtcatgcc atgctgttaa agtaccccgt
      181 tcgaaacact gttcactgtg tcggcgatgc attccattgc aagatcatca ctgcatttgg
      241 atcaacaatt gtgttggcta cggtaactat gaatacttct atcccttcct actagcgaac
      301 tgtaccattt tatcgtatgc atccatcaga ctattttcaa tgttgagagt tctgcgagtt
      361 gtgagcaaac atttcctgtc tctctttttg ttaacaacag cgtttgcatt acttgccata
      421 atatttacat actaccagtt aaagctcgtt caagaaggaa tgaccaacaa cgagcaagat
      481 aaatggtacc tcgtacatga acatatgagg gatggaaacc ttttcaagga taacaatggc
      541 tcactgtact acagaaccag ttctggtgtg gaaggaattg atgacacatt ttactctacc
      601 aacttgtacg atcacagaaa gtacaaactc aacaatcctt cgagaataca cagcgcggaa
      661 gaactcacaa atatttatga tagaggagac ttctggaaga accttgtaga gaggataagt
      721 gtcctttgaa aatctgactt taagaattct cgtagctcca tgtacataat aaatatagac
      781 taataagatt cgcataacaa actggcttca aatcataatg gcaaagaatc acaaaattcg
      841 agatttcaat ctagctaata ttcctaaccg gaattcgcat ctagactgat gagacgaacg
      901 tgagaccgaa agtgaaacgt gatttcatgc gtcattttga acattttgta aatcttattt
      961 aataatgtgt gcggcaattc acatttaatt tatgaatgtt ttcttaacat cgcggcaact
     1021 caagaaacgg caggttcgga tcttagctac tagagaaaga ggagaaatac tagatgcgta
     1081 aaggcgaaga gctgttcact ggtgtcgtcc ctattctggt ggaactggat ggtgatgtca
     1141 acggtcataa gttttccgtg cgtggcgagg gtgaaggtga cgcaactaat ggtaaactga
     1201 cgctgaagtt catctgtact actggtaaac tgccggttcc ttggccgact ctggtaacga
     1261 cgctgactta tggtgttcag tgctttgctc gttatccgga ccatatgaag cagcatgact
     1321 tcttcaagtc cgccatgccg gaaggctatg tgcaggaacg cacgatttcc tttaaggatg
     1381 acggcacgta caaaacgcgt gcggaagtga aatttgaagg cgataccctg gtaaaccgca
     1441 ttgagctgaa aggcattgac tttaaagagg acggcaatat cctgggccat aagctggaat
     1501 acaattttaa cagccacaat gtttacatca ccgccgataa acaaaaaaat ggcattaaag
     1561 cgaattttaa aattcgccac aacgtggagg atggcagcgt gcagctggct gatcactacc
     1621 agcaaaacac tccaatcggt gatggtcctg ttctgctgcc agacaatcac tatctgagca
     1681 cgcaaagcgt tctgtctaaa gatccgaacg agaaacgcga tcatatggtt ctgctggagt
     1741 tcgtaaccgc agcgggcatc acgcatggta tggatgaact gtacaaatga ccaggcatca
     1801 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     1861 gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct gcgtttatag
     1921 gtctcagctg gaaatctgct cgtcagtggt gctcacactg acgaatcatg tacagatcat
     1981 accgatgact gcctggcgac tcacaactaa gcaagacagc cggaaccagc gccggcgaac
     2041 accactgcat atatggcata tcacaacagt ccacgtctca agcagttaca gagatgttac
     2101 gaaccactag tgcactgcag tacatcgagg agaacttcta gtatatctac atacctaata
     2161 ttattgcctt attaaaaatg gaatcccaac aattacatca aaatccacat tctcttcaaa
     2221 atcaattgtc ctgtacttcc ttgttcatgt gtgttcaaaa acgttatatt tataggataa
     2281 ttatactcta tttctcaaca agtaattggt tgtttggccg agcggtctaa ggcgcctgat
     2341 tcaagaaata tcttgaccgc agttaactgt gggaatactc aggtatcgta agatgcaaga
     2401 gttcgaatct cttagcaacc attatttttt tcctcaacat aacgagaaca cacaggggcg
     2461 ctatcgcaca gaatcaaatt cgatgactgg aaattttttg ttaatttcag aggtcgcctg
     2521 acgcatatac ctttttcaac tgaaaaattg ggagaaaaag gaaaggtgag agcgccggaa
     2581 ccggcttttc atatagaata gagaagcgtt catgactaaa tgcttgcatc acaatacttg
     2641 aagttgacaa tattatttaa ggacctattg ttttttccaa taggtggtta gcaatcgtct
     2701 tactttctaa cttttcttac cttttacatt tcagcaatat atatatatat atttcaagga
     2761 tataccattc taatgtctgc ccctaagaag atcgtcgttt tgccaggtga ccacgttggt
     2821 caagaaatca cagccgaagc cattaaggtt cttaaagcta tttctgatgt tcgttccaat
     2881 gtcaagttcg atttcgaaaa tcatttaatt ggtggtgctg ctatcgatgc tacaggtgtt
     2941 ccacttccag atgaggcgct ggaagcctcc aagaaggctg atgccgtttt gttaggtgct
     3001 gtgggtggtc ctaaatgggg aaccggtagt gttagacctg aacaaggttt actaaaaatc
     3061 cgtaaagaac ttcaattgta cgccaactta agaccatgta actttgcatc cgactctctt
     3121 ttagacttat ctccaatcaa gccacaattt gctaaaggta ctgacttcgt tgttgtcaga
     3181 gaattagtgg gaggtattta ctttggtaag agaaaggagg acgatggtga tggtgtcgct
     3241 tgggatagtg aacaatacac cgttccagaa gtgcaaagaa tcacaagaat ggccgctttc
     3301 atggccctac aacatgagcc accattgcct atttggtcct tggataaagc taatgttttg
     3361 gcctcttcaa gattatggag aaaaactgtg gaggaaacca tcaagaacga atttcctaca
     3421 ttgaaggttc aacatcaatt gattgattct gccgccatga tcctagttaa gaacccaacc
     3481 cacctaaatg gtattataat caccagcaac atgtttggtg atatcatctc cgatgaagcc
     3541 tccgttatcc caggttcctt gggtttgttg ccatctgcgt ccttggcctc tttgccagac
     3601 aagaacaccg catttggttt gtacgaacca tgccacggtt ctgctccaga tttgccaaag
     3661 aataaggtca accctatcgc cactatcttg tctgctgcaa tgatgttgaa attgtcattg
     3721 aacttgcctg aagaaggtaa ggccattgaa gatgcagtta aaaaggtttt ggatgcaggt
     3781 atcagaactg gtgatttagg tggttccaac agtaccaccg aagtcggtga tgctgtcgcc
     3841 gaagaagtta agaaaatcct tgcttaacct ggaggaccct tctctttaga ctattctact
     3901 cttatgcacg taaaaaattc taggaaatat gtattaacta ggagtaaaat aaccggctag
     3961 tggcattcat atagccgtct gtttacatct acatcacaca tttcgagtgt atatctcgca
     4021 acgttggcgt taaataggca ggagtaattc aaggttgttc aatattccga ttgttagtag
     4081 attcagttct accttcgctc agttttctta atgactcaaa ggatattaga attgaaaaat
     4141 ttcaaaaaag ctttaagcat agaaccacgc acttattaaa tacaatggag aaatgatttg
     4201 gtgggcaatt accggtttca attgactccg aacaggttca aatacaccac atttaacaga
     4261 ctactaagaa agagtcaaaa agatttggct gaacattaag ggaggctagg ttttttagtg
     4321 actttttgcg agagaaacgc ttttttagca gattaagcaa taagtttaaa catgtccgtt
     4381 gaattgtcca aagtatccat cttgggtcag gacaccattc atgtcggata ccagattcag
     4441 gaacacattg tgaagactac tctggaacaa tgcaaatcgt cgacatatgt tatcatcaac
     4501 gatactaatg tctaccaggt gccttactac catgacttgg ttgagctatt tgagaagtct
     4561 ctacccgaga cttcccgttt gctcacatat gttgtgaagc caggcgaggc taacaagacc
     4621 cgtgaaacta aggcacagat tgaagacttt atgttgaatg aagggtgcac tcgtgatacc
     4681 gtcgttattg caattggtgg tggtgtgatc ggtgatatga ttgggtttgt tgctgcgaca
     4741 ttcatgagag gtgttcgttt cattcaaatt cctacatcgc tattggcaat ggttgactcg
     4801 tctattggtg gtaagactgc cgttgacacc ccaatcggta aaaacttcat tggtgccttc
     4861 tggcaaccac aatttgtgtt ggcgcgcctc agccgagcgg ccgcgtgtta caaccaatta
     4921 accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     4981 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     5041 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     5101 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     5161 tgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcatttcttt ccagacttgt
     5221 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     5281 attcgtgatt gcgcctgagc gaggcgaaat acgcgatcgc tgttaaaagg acaattacaa
     5341 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     5401 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     5461 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     5521 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     5581 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     5641 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     5701 tttaatcgcg gcctggagca agacgtttcc cgttgaatat ggctcataac accccttgta
     5761 ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca
     5821 atgtaacatc agagattttg agacacaacg tggctttgtt gaataaatcg aacttttgct
     5881 gagttgaagg atcagtcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5941 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6001 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6061 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6121 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6181 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6241 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6301 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6361 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6421 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6481 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6541 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6601 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgtg
     6661 c
//
