LOCUS       sequence-317865-        5803 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting integration site I3 
            described in (Rajkumar et al.,2019). Uracil selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5803)
  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 5803)
  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-URA
FEATURES             Location/Qualifiers
     source          1..5803
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1082..1093
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1100..1813
                     /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      1825..1896
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1912..1939
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             2328..3131
                     /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(4100..4915)
                     /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(4100..4915)
                     /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     4823..4842
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4907..4926
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5140..5728
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5629..5648
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcccctgatt ctgtggataa ccgtgcggcc gcccctttca ggcgcgcctc ataccgttta
       61 tgattgcatt tacgttactg accggagttt tgtccgtatt tgtgaagccg ttggagataa
      121 ttgaggcacc aaggttcgaa ccagactatg ctatattcta cccaggaatt aattgccagt
      181 catgccatgc tgttaaagta ccccgttcga aacactgttc actgtgtcgg cgatgcattc
      241 cattgcaaga tcatcactgc atttggatca acaattgtgt tggctacggt aactatgaat
      301 acttctatcc cttcctacta gcgaactgta ccattttatc gtatgcatcc atcagactat
      361 tttcaatgtt gagagttctg cgagttgtga gcaaacattt cctgtctctc tttttgttaa
      421 caacagcgtt tgcattactt gccataatat ttacatacta ccagttaaag ctcgttcaag
      481 aaggaatgac caacaacgag caagataaat ggtacctcgt acatgaacat atgagggatg
      541 gaaacctttt caaggataac aatggctcac tgtactacag aaccagttct ggtgtggaag
      601 gaattgatga cacattttac tctaccaact tgtacgatca cagaaagtac aaactcaaca
      661 atccttcgag aatacacagc gcggaagaac tcacaaatat ttatgataga ggagacttct
      721 ggaagaacct tgtagagagg ataagtgtcc tttgaaaatc tgactttaag aattctcgta
      781 gctccatgta cataataaat atagactaat aagattcgca taacaaactg gcttcaaatc
      841 ataatggcaa agaatcacaa aattcgagat ttcaatctag ctaatattcc taaccggaat
      901 tcgcatctag actgatgaga cgaacgtgag accgaaagtg aaacgtgatt tcatgcgtca
      961 ttttgaacat tttgtaaatc ttatttaata atgtgtgcgg caattcacat ttaatttatg
     1021 aatgttttct taacatcgcg gcaactcaag aaacggcagg ttcggatctt agctactaga
     1081 gaaagaggag aaatactaga tgcgtaaagg cgaagagctg ttcactggtg tcgtccctat
     1141 tctggtggaa ctggatggtg atgtcaacgg tcataagttt tccgtgcgtg gcgagggtga
     1201 aggtgacgca actaatggta aactgacgct gaagttcatc tgtactactg gtaaactgcc
     1261 ggttccttgg ccgactctgg taacgacgct gacttatggt gttcagtgct ttgctcgtta
     1321 tccggaccat atgaagcagc atgacttctt caagtccgcc atgccggaag gctatgtgca
     1381 ggaacgcacg atttccttta aggatgacgg cacgtacaaa acgcgtgcgg aagtgaaatt
     1441 tgaaggcgat accctggtaa accgcattga gctgaaaggc attgacttta aagaggacgg
     1501 caatatcctg ggccataagc tggaatacaa ttttaacagc cacaatgttt acatcaccgc
     1561 cgataaacaa aaaaatggca ttaaagcgaa ttttaaaatt cgccacaacg tggaggatgg
     1621 cagcgtgcag ctggctgatc actaccagca aaacactcca atcggtgatg gtcctgttct
     1681 gctgccagac aatcactatc tgagcacgca aagcgttctg tctaaagatc cgaacgagaa
     1741 acgcgatcat atggttctgc tggagttcgt aaccgcagcg ggcatcacgc atggtatgga
     1801 tgaactgtac aaatgaccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     1861 tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct
     1921 tcgggtgggc ctttctgcgt ttataggtct cagctggaaa tctgctcgtc agtggtgctc
     1981 acactgacga atcatgtaca gatcataccg atgactgcct ggcgactcac aactaagcaa
     2041 gacagccgga accagcgccg gcgaacacca ctgcatatat ggcatatcac aacagtccac
     2101 gtctcaagca gttacagaga tgttacgaac cactagtgca ctgcagtaca cggtttcctt
     2161 gaaatttttt tgattcggta atctccgaac agaaggaaga acgaaggaag gagcacagac
     2221 ttagattggt atatatacgc atatgtagtg ttgaagaaac atgaaattgc ccagtattct
     2281 taacccaact gcacagaaca aaaacgtgca ggaaacgaag ataaatcatg tcgaaagcta
     2341 catataagga acgtgctgct actcatccta gtcctgttgc tgccaagcta tttaatatca
     2401 tgcacgaaaa gcaaacaaac ttgtgtgctt cattggatgt tcgtaccacc aaggaattac
     2461 tggagttagt tgaagcatta ggtcccaaaa tttgtttact aaaaacacat gtggatatct
     2521 tgactgattt ttccatggag ggcacagtta agccgctaaa ggcattatcc gccaagtaca
     2581 attttttact cttcgaggac agaaaatttg ctgacattgg taatacagtc aaattgcagt
     2641 actctgcggg tgtatacaga atagcagaat gggcagacat tacgaatgca cacggtgtgg
     2701 tgggcccagg tattgttagc ggtttgaagc aggcggcaga agaagtaaca aaggaaccta
     2761 gaggcctttt gatgttagca gaattgtcat gcaagggctc cctatctact ggagaatata
     2821 ctaagggtac tgttgacatt gcgaagagcg acaaagattt tgttatcggc tttattgctc
     2881 aaagagacat gggtggaaga gatgaaggtt acgattggtt gattatgaca cccggtgtgg
     2941 gtttagatga caagggagat gcattgggtc aacagtatag aaccgtggat gatgtggttt
     3001 ctacaggatc tgacattatt attgttggaa gaggactatt tgcaaaggga agggatgcta
     3061 aggtagaggg tgaacgttac agaaaagcag gctgggaagc atatttgaga agatgcggcc
     3121 agcaaaacta aaaaactgta ttataagtaa atgcatgtat actaaactca caaattagag
     3181 cttcaattta attatatcag ttattacccg agtaattcaa ggttgttcaa tattccgatt
     3241 gttagtagat tcagttctac cttcgctcag ttttcttaat gactcaaagg atattagaat
     3301 tgaaaaattt caaaaaagct ttaagcatag aaccacgcac ttattaaata caatggagaa
     3361 atgatttggt gggcaattac cggtttcaat tgactccgaa caggttcaaa tacaccacat
     3421 ttaacagact actaagaaag agtcaaaaag atttggctga acattaaggg aggctaggtt
     3481 ttttagtgac tttttgcgag agaaacgctt ttttagcaga ttaagcaata agtttaaaca
     3541 tgtccgttga attgtccaaa gtatccatct tgggtcagga caccattcat gtcggatacc
     3601 agattcagga acacattgtg aagactactc tggaacaatg caaatcgtcg acatatgtta
     3661 tcatcaacga tactaatgtc taccaggtgc cttactacca tgacttggtt gagctatttg
     3721 agaagtctct acccgagact tcccgtttgc tcacatatgt tgtgaagcca ggcgaggcta
     3781 acaagacccg tgaaactaag gcacagattg aagactttat gttgaatgaa gggtgcactc
     3841 gtgataccgt cgttattgca attggtggtg gtgtgatcgg tgatatgatt gggtttgttg
     3901 ctgcgacatt catgagaggt gttcgtttca ttcaaattcc tacatcgcta ttggcaatgg
     3961 ttgactcgtc tattggtggt aagactgccg ttgacacccc aatcggtaaa aacttcattg
     4021 gtgccttctg gcaaccacaa tttgtgttgg cgcgcctcag ccgagcggcc gcgtgttaca
     4081 accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat
     4141 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     4201 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     4261 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     4321 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc
     4381 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     4441 cgttattcat tcgtgattgc gcctgagcga ggcgaaatac gcgatcgctg ttaaaaggac
     4501 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     4561 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     4621 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     4681 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     4741 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     4801 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     4861 tgttggaatt taatcgcggc ctggagcaag acgtttcccg ttgaatatgg ctcataacac
     4921 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat
     4981 cttgtgcaat gtaacatcag agattttgag acacaacgtg gctttgttga ataaatcgaa
     5041 cttttgctga gttgaaggat cagtcatgac caaaatccct taacgtgagt tttcgttcca
     5101 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5161 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5221 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5281 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5341 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5401 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5461 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5521 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5581 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5641 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5701 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5761 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg tta
//
