LOCUS       sequence-318043-        6679 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting integration site I4 
            described in (Rajkumar et al.,2019). Leucine selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6679)
  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 6679)
  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: pI4-MTU-DO-LEU
FEATURES             Location/Qualifiers
     source          1..6679
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1086..1097
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1104..1817
                     /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      1829..1900
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1916..1943
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        2395..2802
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2803..3897
                     /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(4978..5793)
                     /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(4978..5793)
                     /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     5701..5720
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5785..5804
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6018..6606
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6507..6526
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tatcccctga ttctgtggat aaccgtgcgg ccgccccttt caggcgcgcc ggtgttgcat
       61 tagtatcgcc gtggatgaag atgaaggtgg aatagacgtt acccatagat acccgaatga
      121 gtatctgatt gaggattttt gacttactta cttcttcgag atacactact ctgccgaaat
      181 ttcggactgg aatgcgtcaa agtgaatcca ctttgcagga aaacgcagga aaaacggatt
      241 cgaaaacgca acaaaaaaaa aaaaattata cataataaaa tttgaatcaa agcaacaagt
      301 tcacgaatcc attaaagcag tcagaatcca ggaaaaaaaa gaaacagagc cagaaaaaag
      361 gcagcaacga atcacaacca cagcgtattg cgtcagtgga aattaattac cttggtattt
      421 ccaatgctgt gtacaaccaa aatttccaca tagttagttg attcgcattt ccctggtaac
      481 cgccctcggc aacctccctc ggaaacttcc ctccagagta acttccccca gagttataga
      541 gatatagagt tatagtagag atatagagct atagatagat tgattcttgt tgtagcattt
      601 attccgttaa acttctgcgg ctaattttgc ctagcttggc cttttttttt tgtttttcga
      661 gctttcaaag acaaatggac attcttacat aatatattat catatacacc gagatcggtg
      721 ggtcagagcc tgtagggatg gacattttcc ttctagactc gccttccaga cctttttttg
      781 gtttgtttct ctctccgtgg aactcgcttt ctttttcgcc ggtgatctcg aaacgtaatc
      841 gatatgtatt ccattccatt cgctattttt tttgttccat aaaccatgtt cttatgaaca
      901 gaattcgcat ctagactgat gagacgaacg tgagaccgaa agtgaaacgt gatttcatgc
      961 gtcattttga acattttgta aatcttattt aataatgtgt gcggcaattc acatttaatt
     1021 tatgaatgtt ttcttaacat cgcggcaact caagaaacgg caggttcgga tcttagctac
     1081 tagagaaaga ggagaaatac tagatgcgta aaggcgaaga gctgttcact ggtgtcgtcc
     1141 ctattctggt ggaactggat ggtgatgtca acggtcataa gttttccgtg cgtggcgagg
     1201 gtgaaggtga cgcaactaat ggtaaactga cgctgaagtt catctgtact actggtaaac
     1261 tgccggttcc ttggccgact ctggtaacga cgctgactta tggtgttcag tgctttgctc
     1321 gttatccgga ccatatgaag cagcatgact tcttcaagtc cgccatgccg gaaggctatg
     1381 tgcaggaacg cacgatttcc tttaaggatg acggcacgta caaaacgcgt gcggaagtga
     1441 aatttgaagg cgataccctg gtaaaccgca ttgagctgaa aggcattgac tttaaagagg
     1501 acggcaatat cctgggccat aagctggaat acaattttaa cagccacaat gtttacatca
     1561 ccgccgataa acaaaaaaat ggcattaaag cgaattttaa aattcgccac aacgtggagg
     1621 atggcagcgt gcagctggct gatcactacc agcaaaacac tccaatcggt gatggtcctg
     1681 ttctgctgcc agacaatcac tatctgagca cgcaaagcgt tctgtctaaa gatccgaacg
     1741 agaaacgcga tcatatggtt ctgctggagt tcgtaaccgc agcgggcatc acgcatggta
     1801 tggatgaact gtacaaatga ccaggcatca aataaaacga aaggctcagt cgaaagactg
     1861 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc acactggctc
     1921 accttcgggt gggcctttct gcgtttatag gtctcagctg gaaatctgct cgtcagtggt
     1981 gctcacactg acgaatcatg tacagatcat accgatgact gcctggcgac tcacaactaa
     2041 gcaagacagc cggaaccagc gccggcgaac accactgcat atatggcata tcacaacagt
     2101 ccacgtctca agcagttaca gagatgttac gaaccactag tgcactgcag tacatcgagg
     2161 agaacttcta gtatatctac atacctaata ttattgcctt attaaaaatg gaatcccaac
     2221 aattacatca aaatccacat tctcttcaaa atcaattgtc ctgtacttcc ttgttcatgt
     2281 gtgttcaaaa acgttatatt tataggataa ttatactcta tttctcaaca agtaattggt
     2341 tgtttggccg agcggtctaa ggcgcctgat tcaagaaata tcttgaccgc agttaactgt
     2401 gggaatactc aggtatcgta agatgcaaga gttcgaatct cttagcaacc attatttttt
     2461 tcctcaacat aacgagaaca cacaggggcg ctatcgcaca gaatcaaatt cgatgactgg
     2521 aaattttttg ttaatttcag aggtcgcctg acgcatatac ctttttcaac tgaaaaattg
     2581 ggagaaaaag gaaaggtgag agcgccggaa ccggcttttc atatagaata gagaagcgtt
     2641 catgactaaa tgcttgcatc acaatacttg aagttgacaa tattatttaa ggacctattg
     2701 ttttttccaa taggtggtta gcaatcgtct tactttctaa cttttcttac cttttacatt
     2761 tcagcaatat atatatatat atttcaagga tataccattc taatgtctgc ccctaagaag
     2821 atcgtcgttt tgccaggtga ccacgttggt caagaaatca cagccgaagc cattaaggtt
     2881 cttaaagcta tttctgatgt tcgttccaat gtcaagttcg atttcgaaaa tcatttaatt
     2941 ggtggtgctg ctatcgatgc tacaggtgtt ccacttccag atgaggcgct ggaagcctcc
     3001 aagaaggctg atgccgtttt gttaggtgct gtgggtggtc ctaaatgggg aaccggtagt
     3061 gttagacctg aacaaggttt actaaaaatc cgtaaagaac ttcaattgta cgccaactta
     3121 agaccatgta actttgcatc cgactctctt ttagacttat ctccaatcaa gccacaattt
     3181 gctaaaggta ctgacttcgt tgttgtcaga gaattagtgg gaggtattta ctttggtaag
     3241 agaaaggagg acgatggtga tggtgtcgct tgggatagtg aacaatacac cgttccagaa
     3301 gtgcaaagaa tcacaagaat ggccgctttc atggccctac aacatgagcc accattgcct
     3361 atttggtcct tggataaagc taatgttttg gcctcttcaa gattatggag aaaaactgtg
     3421 gaggaaacca tcaagaacga atttcctaca ttgaaggttc aacatcaatt gattgattct
     3481 gccgccatga tcctagttaa gaacccaacc cacctaaatg gtattataat caccagcaac
     3541 atgtttggtg atatcatctc cgatgaagcc tccgttatcc caggttcctt gggtttgttg
     3601 ccatctgcgt ccttggcctc tttgccagac aagaacaccg catttggttt gtacgaacca
     3661 tgccacggtt ctgctccaga tttgccaaag aataaggtca accctatcgc cactatcttg
     3721 tctgctgcaa tgatgttgaa attgtcattg aacttgcctg aagaaggtaa ggccattgaa
     3781 gatgcagtta aaaaggtttt ggatgcaggt atcagaactg gtgatttagg tggttccaac
     3841 agtaccaccg aagtcggtga tgctgtcgcc gaagaagtta agaaaatcct tgcttaacct
     3901 ggaggaccct tctctttaga ctattctact cttatgcacg taaaaaattc taggaaatat
     3961 gtattaacta ggagtaaaat aaccggctag tggcattcat atagccgtct gtttacatct
     4021 acatcacaca tttcgagtgt atatctcgca acgttggcgt taaataggca ggagtttcac
     4081 aatggaggga gtgaaatcta aaagtggaaa aaaaaagggg aggcgaaaaa caaaaaaaaa
     4141 aaaaagaacg aaccgaacta tgtccacagg agcagaagca gaagctgcgg cagcagcttc
     4201 tgcggctttg aaagaggaaa cgcgtagatg aaagaatgat ccaagcccgg ctttccctct
     4261 gtgcgcggat tgttattttt ttagacttat ttctatgttt gttttggacc taatataact
     4321 tagtcatgaa tcctggttga ctcgcgcgca tctttttcca ctctgcgatg attcacagtg
     4381 cagcgtgcag ctcagctcca gcagcagtac cagcggcagc tccaacagtg gagccttcct
     4441 ttcacgaacg gaatagaaac aggaaggaaa aaaatagacc aacaagagga cccatccgcg
     4501 acttctaatt tttggcgcat gaaccgcgct gttctacccg cccgtccgtc ctcatccctc
     4561 ccccccagga aaacaggaaa ttgggaaacc taggtgcgtg cattttcgtt ttgggcgatg
     4621 attgacaatg agacttagtc cgcttggttg ggtttcgttt caaaaacaag gtttggaaga
     4681 agatcttggg agagaaagac cgagagatct tgaccagggc gaagacgacc agtagctttt
     4741 ggtatagccg ggataagctt caggcaccct agggttgtat aagagttgta tgcaaggatg
     4801 tgcgcaaggg tttctgaagc aggatggaaa tgtggtgtgt ttttttgtag agcaattgcc
     4861 ttaaagtcgg ggattacgcc gagacagcag cttgacggtt ccgtggtaat atgtgtccgg
     4921 caacttggcg cgcctcagcc gagcggccgc gtgttacaac caattaacca attctgatta
     4981 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     5041 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     5101 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     5161 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     5221 atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa caggccagcc
     5281 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     5341 ctgagcgagg cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     5401 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     5461 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     5521 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     5581 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     5641 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     5701 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     5761 ggagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     5821 agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt aacatcagag
     5881 attttgagac acaacgtggc tttgttgaat aaatcgaact tttgctgagt tgaaggatca
     5941 gtcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6001 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6061 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6121 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6181 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6241 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6301 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6361 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6421 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6481 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     6541 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     6601 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     6661 cacatgttct ttcctgcgt
//
