LOCUS       sequence-317861-        6972 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting integration site I2 
            described in (Rajkumar et al.,2019). Leucine selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6972)
  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 6972)
  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-LEU
FEATURES             Location/Qualifiers
     source          1..6972
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1198..1209
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1216..1929
                     /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      1941..2012
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2028..2055
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        2507..2914
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2915..4009
                     /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(5234..6049)
                     /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(5234..6049)
                     /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     5957..5976
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6041..6060
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6274..6862
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6763..6782
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttcaggcgc gccaccgaat attaaataat ctttttttat ttcttatgta cgcctgcttt
       61 gtatgataca aaatcatcgt atcgtattcg gcttcatgtt atccgtcgaa tttagattac
      121 cttggctaag gggccaaggt ggatgattcg tatgagtatc acaatttaca aattacaagt
      181 tttgcaaaga gtatgtaaac tgtaaaaaag aaaacattga caaatatacc tgaaaagagt
      241 ccattttcta catttgagaa aaaaaggaat aaagtgattc tcctcatcgc tcataaaaaa
      301 atactattca tgagcaaaat tgagaaaaat ggcaatcaca ttcaatacta gttagatagc
      361 catatctaaa ctgctgtacg tggtttaata ctagcaccaa catcatttga ataatggcga
      421 agaaaccaat ttcgaaacat tcccgtgcgg caagaagatc agaagcagcc gaaccggagg
      481 ctaaggcttt agagacttta cccagagctg aaaagacaga ttttgcagca acattaatca
      541 gaactgctaa caaaaacgaa gctttgctcg aagcgaagat ccaaaagcgt ggacaaaaaa
      601 ataagaagaa aaacgcggga agagtgagta agcaacttat agatgacagt tctttaacaa
      661 gtaggaaagc tattcgtgcc ttaaatatta ccagtaggct cgatggtaag agggagaaag
      721 ccattcatag agctaaatat gtgcagtcag ctcgtaaagc tggatgggat actacaaacc
      781 aaagcattag gaaagaatta tcgacactac aaaacgcagc aaatacggtc agcgattccg
      841 ttaacaaaag cacggacaag gaagggtctg cgaatactga agatgcgatg atagaagacg
      901 atcaagatca cattgaaatt tacgacagtg ctaaaagcca gaaggtacaa gaaaaagcag
      961 tatcaaccac ttttacggca tcaaacgtat ttacgtcatt agaggatgag atgaattcgc
     1021 atctagactg atgagacgaa cgtgagaccg aaagtgaaac gtgatttcat gcgtcatttt
     1081 gaacattttg taaatcttat ttaataatgt gtgcggcaat tcacatttaa tttatgaatg
     1141 ttttcttaac atcgcggcaa ctcaagaaac ggcaggttcg gatcttagct actagagaaa
     1201 gaggagaaat actagatgcg taaaggcgaa gagctgttca ctggtgtcgt ccctattctg
     1261 gtggaactgg atggtgatgt caacggtcat aagttttccg tgcgtggcga gggtgaaggt
     1321 gacgcaacta atggtaaact gacgctgaag ttcatctgta ctactggtaa actgccggtt
     1381 ccttggccga ctctggtaac gacgctgact tatggtgttc agtgctttgc tcgttatccg
     1441 gaccatatga agcagcatga cttcttcaag tccgccatgc cggaaggcta tgtgcaggaa
     1501 cgcacgattt cctttaagga tgacggcacg tacaaaacgc gtgcggaagt gaaatttgaa
     1561 ggcgataccc tggtaaaccg cattgagctg aaaggcattg actttaaaga ggacggcaat
     1621 atcctgggcc ataagctgga atacaatttt aacagccaca atgtttacat caccgccgat
     1681 aaacaaaaaa atggcattaa agcgaatttt aaaattcgcc acaacgtgga ggatggcagc
     1741 gtgcagctgg ctgatcacta ccagcaaaac actccaatcg gtgatggtcc tgttctgctg
     1801 ccagacaatc actatctgag cacgcaaagc gttctgtcta aagatccgaa cgagaaacgc
     1861 gatcatatgg ttctgctgga gttcgtaacc gcagcgggca tcacgcatgg tatggatgaa
     1921 ctgtacaaat gaccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     1981 gttttatctg ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg
     2041 gtgggccttt ctgcgtttat aggtctcagc tggaaatctg ctcgtcagtg gtgctcacac
     2101 tgacgaatca tgtacagatc ataccgatga ctgcctggcg actcacaact aagcaagaca
     2161 gccggaacca gcgccggcga acaccactgc atatatggca tatcacaaca gtccacgtct
     2221 caagcagtta cagagatgtt acgaaccact agtgcactgc agtacatcga ggagaacttc
     2281 tagtatatct acatacctaa tattattgcc ttattaaaaa tggaatccca acaattacat
     2341 caaaatccac attctcttca aaatcaattg tcctgtactt ccttgttcat gtgtgttcaa
     2401 aaacgttata tttataggat aattatactc tatttctcaa caagtaattg gttgtttggc
     2461 cgagcggtct aaggcgcctg attcaagaaa tatcttgacc gcagttaact gtgggaatac
     2521 tcaggtatcg taagatgcaa gagttcgaat ctcttagcaa ccattatttt tttcctcaac
     2581 ataacgagaa cacacagggg cgctatcgca cagaatcaaa ttcgatgact ggaaattttt
     2641 tgttaatttc agaggtcgcc tgacgcatat acctttttca actgaaaaat tgggagaaaa
     2701 aggaaaggtg agagcgccgg aaccggcttt tcatatagaa tagagaagcg ttcatgacta
     2761 aatgcttgca tcacaatact tgaagttgac aatattattt aaggacctat tgttttttcc
     2821 aataggtggt tagcaatcgt cttactttct aacttttctt accttttaca tttcagcaat
     2881 atatatatat atatttcaag gatataccat tctaatgtct gcccctaaga agatcgtcgt
     2941 tttgccaggt gaccacgttg gtcaagaaat cacagccgaa gccattaagg ttcttaaagc
     3001 tatttctgat gttcgttcca atgtcaagtt cgatttcgaa aatcatttaa ttggtggtgc
     3061 tgctatcgat gctacaggtg ttccacttcc agatgaggcg ctggaagcct ccaagaaggc
     3121 tgatgccgtt ttgttaggtg ctgtgggtgg tcctaaatgg ggaaccggta gtgttagacc
     3181 tgaacaaggt ttactaaaaa tccgtaaaga acttcaattg tacgccaact taagaccatg
     3241 taactttgca tccgactctc ttttagactt atctccaatc aagccacaat ttgctaaagg
     3301 tactgacttc gttgttgtca gagaattagt gggaggtatt tactttggta agagaaagga
     3361 ggacgatggt gatggtgtcg cttgggatag tgaacaatac accgttccag aagtgcaaag
     3421 aatcacaaga atggccgctt tcatggccct acaacatgag ccaccattgc ctatttggtc
     3481 cttggataaa gctaatgttt tggcctcttc aagattatgg agaaaaactg tggaggaaac
     3541 catcaagaac gaatttccta cattgaaggt tcaacatcaa ttgattgatt ctgccgccat
     3601 gatcctagtt aagaacccaa cccacctaaa tggtattata atcaccagca acatgtttgg
     3661 tgatatcatc tccgatgaag cctccgttat cccaggttcc ttgggtttgt tgccatctgc
     3721 gtccttggcc tctttgccag acaagaacac cgcatttggt ttgtacgaac catgccacgg
     3781 ttctgctcca gatttgccaa agaataaggt caaccctatc gccactatct tgtctgctgc
     3841 aatgatgttg aaattgtcat tgaacttgcc tgaagaaggt aaggccattg aagatgcagt
     3901 taaaaaggtt ttggatgcag gtatcagaac tggtgattta ggtggttcca acagtaccac
     3961 cgaagtcggt gatgctgtcg ccgaagaagt taagaaaatc cttgcttaac ctggaggacc
     4021 cttctcttta gactattcta ctcttatgca cgtaaaaaat tctaggaaat atgtattaac
     4081 taggagtaaa ataaccggct agtggcattc atatagccgt ctgtttacat ctacatcaca
     4141 catttcgagt gtatatctcg caacgttggc gttaaatagg caggagttga agcatagaag
     4201 aaatgtttaa ccaaaaacta tcctttttat ttattttgaa ttttctctct catatttttg
     4261 taaaacgata ctttaaccac actcaagctt ttgcttgcta ctcaaatacc tgaaatcttt
     4321 atagacaatt ttccttctta ttcgtaataa taaaactata ttgttctctt cctgtttttt
     4381 tttttaaaaa aaaaaactgt caaaatctcc cccacagaaa aataaaaaca aattatactt
     4441 ttgggagtaa accatgaact taaaacaaca gttcatattc ttaagtagtg ccactatagt
     4501 taaccagaga caatttaact cctaaatctt catactgaag attgacataa actaaataat
     4561 gactacactg ttacttttga gtcagttggg tcttttggca ttcgccaatg ctattaaccc
     4621 tatggtaaag ggcgaatata gaattgaaca tcctaagata ggagacttgt cagaaattga
     4681 atctgctatc tctcccaaat ctttctatga atcatttcaa tatggttccg aagcggagtt
     4741 ccgtaagaat tgggtttctt ctgatctaac aatggaacat gtagacgaaa catcttcgaa
     4801 ggcgttccct ggcacatgga agttgcagtc accatttttg gtcccaggat tcagtgatga
     4861 tctatcttta gttttgggta gcgaacaaac tagagctgct attgcccata ggttaaaata
     4921 cgatctatcg atcaaggata atgagaaact agtggtgcaa tatgaagtta aacttcaaaa
     4981 aatgcttgaa tgtggcggtg catatatcaa attgttgaaa acctccgcaa ctgatctttc
     5041 tcagtacgac cactcgtcag atgactatgc tgtcatcttt ggcccagact tttgccaagg
     5101 ttatgtggac gaaattcatt tggttttgaa gcgcgagaat cctttttctg gaaaatctga
     5161 agacaaattc ttattaaatg ccggcgcgcc tcagccgagc ggccgcgtgt tacaaccaat
     5221 taaccaattc tgattagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat
     5281 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
     5341 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa
     5401 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac
     5461 catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct ttccagactt
     5521 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat
     5581 tcattcgtga ttgcgcctga gcgaggcgaa atacgcgatc gctgttaaaa ggacaattac
     5641 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac
     5701 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga
     5761 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt
     5821 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc
     5881 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
     5941 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg
     6001 aatttaatcg cggcctggag caagacgttt cccgttgaat atggctcata acaccccttg
     6061 tattactgtt tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg
     6121 caatgtaaca tcagagattt tgagacacaa cgtggctttg ttgaataaat cgaacttttg
     6181 ctgagttgaa ggatcagtca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6241 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6301 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6361 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6421 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6481 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6541 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6601 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6661 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6721 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6781 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6841 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     6901 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     6961 tgcggccgcc cc
//
