LOCUS       sequence-317073-        5877 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting the LAC4 locus. 
            Uracil selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5877)
  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 5877)
  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: pI1-MTU-DO-URA
FEATURES             Location/Qualifiers
     source          1..5877
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1129..1140
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1147..1860
                     /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      1872..1943
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1959..1986
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             2375..3178
                     /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"
     CDS             complement(4191..5006)
                     /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(4191..5006)
                     /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     4914..4933
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4998..5017
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5231..5819
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5720..5739
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgttctttcc tgcgttatcc cctgattctg tggataaccg tgcggccgcc cctttcaggc
       61 gcgccgaaag tgattgaaga accctcaaac ttgctggata tttccttacc cttgaccttt
      121 aggctttcaa ttttacccaa caatttgtcc aagacaaatt gcaatccact ggattcaact
      181 gagacataac gtttaccgtc gttgatcttg gtaggttttt ctgctgtctc tgtaacaaaa
      241 tctggtacct ttaatggaag ttcggcttgg ccccaggcaa tttcatgacc tgcctttaga
      301 acaccagcat catcctttaa cacggcaaca acataagtgg tatcagaagg aatagtaaca
      361 gattcttctg gctttaaagc tggaacgtcg attgtctttc ccgtgtcctt gtcgataaac
      421 aataagtgat ctgtcgtaat gaagtcgtgc ttatttgtga ttgttacaga tccgtgcgca
      481 attttaatat gaacgggttc aataaccttc ttatactcta caaggcccgg agtaggatta
      541 tgctcactgt tacacaaacc atccatgatg aacactccgt catgaacctc ttccttaaag
      601 tcaccaccat aagcataagc tttatgcaac ttaccatctg cagtactaac atcttcgaat
      661 tcaataccgt gatttgccca ttcccagata aagccacctt ggtaaaactt ctccttgtag
      721 aacaactctt gatattcttt caaagaaccc gggccgttac ccattgcatg accgtactca
      781 cacaagatca aaggcttttc aaacttacca ttttcatcag tgtggttctt cctccacctt
      841 tccataattt caaatgttgg gtacatgaaa ctaaagatat ctgcactcaa agcgttcaag
      901 tcaccctcat aatgcaccag tctggtagga tccaattgct taagaattcg catctagact
      961 gatgagacga acgtgagacc gaaagtgaaa cgtgatttca tgcgtcattt tgaacatttt
     1021 gtaaatctta tttaataatg tgtgcggcaa ttcacattta atttatgaat gttttcttaa
     1081 catcgcggca actcaagaaa cggcaggttc ggatcttagc tactagagaa agaggagaaa
     1141 tactagatgc gtaaaggcga agagctgttc actggtgtcg tccctattct ggtggaactg
     1201 gatggtgatg tcaacggtca taagttttcc gtgcgtggcg agggtgaagg tgacgcaact
     1261 aatggtaaac tgacgctgaa gttcatctgt actactggta aactgccggt tccttggccg
     1321 actctggtaa cgacgctgac ttatggtgtt cagtgctttg ctcgttatcc ggaccatatg
     1381 aagcagcatg acttcttcaa gtccgccatg ccggaaggct atgtgcagga acgcacgatt
     1441 tcctttaagg atgacggcac gtacaaaacg cgtgcggaag tgaaatttga aggcgatacc
     1501 ctggtaaacc gcattgagct gaaaggcatt gactttaaag aggacggcaa tatcctgggc
     1561 cataagctgg aatacaattt taacagccac aatgtttaca tcaccgccga taaacaaaaa
     1621 aatggcatta aagcgaattt taaaattcgc cacaacgtgg aggatggcag cgtgcagctg
     1681 gctgatcact accagcaaaa cactccaatc ggtgatggtc ctgttctgct gccagacaat
     1741 cactatctga gcacgcaaag cgttctgtct aaagatccga acgagaaacg cgatcatatg
     1801 gttctgctgg agttcgtaac cgcagcgggc atcacgcatg gtatggatga actgtacaaa
     1861 tgaccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     1921 gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt
     1981 tctgcgttta taggtctcag ctggaaatct gctcgtcagt ggtgctcaca ctgacgaatc
     2041 atgtacagat cataccgatg actgcctggc gactcacaac taagcaagac agccggaacc
     2101 agcgccggcg aacaccactg catatatggc atatcacaac agtccacgtc tcaagcagtt
     2161 acagagatgt tacgaaccac tagtgcactg cagtacacgg tttccttgaa atttttttga
     2221 ttcggtaatc tccgaacaga aggaagaacg aaggaaggag cacagactta gattggtata
     2281 tatacgcata tgtagtgttg aagaaacatg aaattgccca gtattcttaa cccaactgca
     2341 cagaacaaaa acgtgcagga aacgaagata aatcatgtcg aaagctacat ataaggaacg
     2401 tgctgctact catcctagtc ctgttgctgc caagctattt aatatcatgc acgaaaagca
     2461 aacaaacttg tgtgcttcat tggatgttcg taccaccaag gaattactgg agttagttga
     2521 agcattaggt cccaaaattt gtttactaaa aacacatgtg gatatcttga ctgatttttc
     2581 catggagggc acagttaagc cgctaaaggc attatccgcc aagtacaatt ttttactctt
     2641 cgaggacaga aaatttgctg acattggtaa tacagtcaaa ttgcagtact ctgcgggtgt
     2701 atacagaata gcagaatggg cagacattac gaatgcacac ggtgtggtgg gcccaggtat
     2761 tgttagcggt ttgaagcagg cggcagaaga agtaacaaag gaacctagag gccttttgat
     2821 gttagcagaa ttgtcatgca agggctccct atctactgga gaatatacta agggtactgt
     2881 tgacattgcg aagagcgaca aagattttgt tatcggcttt attgctcaaa gagacatggg
     2941 tggaagagat gaaggttacg attggttgat tatgacaccc ggtgtgggtt tagatgacaa
     3001 gggagatgca ttgggtcaac agtatagaac cgtggatgat gtggtttcta caggatctga
     3061 cattattatt gttggaagag gactatttgc aaagggaagg gatgctaagg tagagggtga
     3121 acgttacaga aaagcaggct gggaagcata tttgagaaga tgcggccagc aaaactaaaa
     3181 aactgtatta taagtaaatg catgtatact aaactcacaa attagagctt caatttaatt
     3241 atatcagtta ttacccgagt tgcttaatta gcttgtacat ggctttgtgg tttctgccat
     3301 aacaagcttc gttacccaag gaccagataa taatcgaagg atgattgaca tctcttagga
     3361 caagttggga agctctgtct aagtacgcga cctcgtactc tggattatct gataagtaat
     3421 gggcattaac atcgtagagt ttatttttgg tatctggata ttcagcctcc aagttcgtat
     3481 gacgattaaa tggctcttga acaccgtgag tttcaagatc tgcctcatca atgacccaga
     3541 agcctagttt atcgaagagg tcatacacct taggatggtt tggataatgc gagttacgaa
     3601 cagcattgat gttaaacttc ttcattagaa tcaagtctct aacaacaaaa tctaatggca
     3661 cagctctacc gaaccttgga tggtgatcat gcctgttgac acctctaaag agaatgtctt
     3721 tgccattaac agtaatgtta ccgtccttca actccacttg tctgaaacca acatggtgct
     3781 taatagattg aatcacactg ccatcagatc caattaaatc caactggtac ttgtacaaag
     3841 taggattttc tgcggtccaa tgttctgggg ccttgacgtt gatcttgaaa gctgtttctt
     3901 cgtccttttt ggtcgaaaag gaaataaatt ctttagttga aaacgtcgtg ttcccattct
     3961 cctcgttcaa caaagagctt gcatcgtaaa ctttagatcc atcttcaggt tcgtaaagtg
     4021 tgaaattgat gtgatcataa gaagaaccct ggacatcaac tttcacagaa agctctgcat
     4081 cctgatactg agagtccaca aaagttgtag tgaccctaac gtcttcaata tgggccttcg
     4141 gcgcgcctca gccgagcggc cgcgtgttac aaccaattaa ccaattctga ttagaaaaac
     4201 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     4261 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     4321 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     4381 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     4441 gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca gccattacgc
     4501 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     4561 aggcgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     4621 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     4681 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     4741 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     4801 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     4861 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     4921 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctggagcaa
     4981 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     5041 agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca gagattttga
     5101 gacacaacgt ggctttgttg aataaatcga acttttgctg agttgaagga tcagtcatga
     5161 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5221 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5281 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     5341 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     5401 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5461 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5521 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     5581 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5641 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5701 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5761 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5821 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcaca
//
