LOCUS       sequence-317966-        5821 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting integration site I4 
            described in (Rajkumar et al.,2019). Uracil selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5821)
  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 5821)
  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-URA
FEATURES             Location/Qualifiers
     source          1..5821
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1038..1049
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1056..1769
                     /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      1781..1852
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1868..1895
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             2284..3087
                     /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(4072..4887)
                     /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(4072..4887)
                     /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     4795..4814
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4879..4898
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5112..5700
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5601..5620
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccggtgttgc attagtatcg ccgtggatga agatgaaggt ggaatagacg ttacccatag
       61 atacccgaat gagtatctga ttgaggattt ttgacttact tacttcttcg agatacacta
      121 ctctgccgaa atttcggact ggaatgcgtc aaagtgaatc cactttgcag gaaaacgcag
      181 gaaaaacgga ttcgaaaacg caacaaaaaa aaaaaaatta tacataataa aatttgaatc
      241 aaagcaacaa gttcacgaat ccattaaagc agtcagaatc caggaaaaaa aagaaacaga
      301 gccagaaaaa aggcagcaac gaatcacaac cacagcgtat tgcgtcagtg gaaattaatt
      361 accttggtat ttccaatgct gtgtacaacc aaaatttcca catagttagt tgattcgcat
      421 ttccctggta accgccctcg gcaacctccc tcggaaactt ccctccagag taacttcccc
      481 cagagttata gagatataga gttatagtag agatatagag ctatagatag attgattctt
      541 gttgtagcat ttattccgtt aaacttctgc ggctaatttt gcctagcttg gccttttttt
      601 tttgtttttc gagctttcaa agacaaatgg acattcttac ataatatatt atcatataca
      661 ccgagatcgg tgggtcagag cctgtaggga tggacatttt ccttctagac tcgccttcca
      721 gacctttttt tggtttgttt ctctctccgt ggaactcgct ttctttttcg ccggtgatct
      781 cgaaacgtaa tcgatatgta ttccattcca ttcgctattt tttttgttcc ataaaccatg
      841 ttcttatgaa cagaattcgc atctagactg atgagacgaa cgtgagaccg aaagtgaaac
      901 gtgatttcat gcgtcatttt gaacattttg taaatcttat ttaataatgt gtgcggcaat
      961 tcacatttaa tttatgaatg ttttcttaac atcgcggcaa ctcaagaaac ggcaggttcg
     1021 gatcttagct actagagaaa gaggagaaat actagatgcg taaaggcgaa gagctgttca
     1081 ctggtgtcgt ccctattctg gtggaactgg atggtgatgt caacggtcat aagttttccg
     1141 tgcgtggcga gggtgaaggt gacgcaacta atggtaaact gacgctgaag ttcatctgta
     1201 ctactggtaa actgccggtt ccttggccga ctctggtaac gacgctgact tatggtgttc
     1261 agtgctttgc tcgttatccg gaccatatga agcagcatga cttcttcaag tccgccatgc
     1321 cggaaggcta tgtgcaggaa cgcacgattt cctttaagga tgacggcacg tacaaaacgc
     1381 gtgcggaagt gaaatttgaa ggcgataccc tggtaaaccg cattgagctg aaaggcattg
     1441 actttaaaga ggacggcaat atcctgggcc ataagctgga atacaatttt aacagccaca
     1501 atgtttacat caccgccgat aaacaaaaaa atggcattaa agcgaatttt aaaattcgcc
     1561 acaacgtgga ggatggcagc gtgcagctgg ctgatcacta ccagcaaaac actccaatcg
     1621 gtgatggtcc tgttctgctg ccagacaatc actatctgag cacgcaaagc gttctgtcta
     1681 aagatccgaa cgagaaacgc gatcatatgg ttctgctgga gttcgtaacc gcagcgggca
     1741 tcacgcatgg tatggatgaa ctgtacaaat gaccaggcat caaataaaac gaaaggctca
     1801 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tctactagag
     1861 tcacactggc tcaccttcgg gtgggccttt ctgcgtttat aggtctcagc tggaaatctg
     1921 ctcgtcagtg gtgctcacac tgacgaatca tgtacagatc ataccgatga ctgcctggcg
     1981 actcacaact aagcaagaca gccggaacca gcgccggcga acaccactgc atatatggca
     2041 tatcacaaca gtccacgtct caagcagtta cagagatgtt acgaaccact agtgcactgc
     2101 agtacacggt ttccttgaaa tttttttgat tcggtaatct ccgaacagaa ggaagaacga
     2161 aggaaggagc acagacttag attggtatat atacgcatat gtagtgttga agaaacatga
     2221 aattgcccag tattcttaac ccaactgcac agaacaaaaa cgtgcaggaa acgaagataa
     2281 atcatgtcga aagctacata taaggaacgt gctgctactc atcctagtcc tgttgctgcc
     2341 aagctattta atatcatgca cgaaaagcaa acaaacttgt gtgcttcatt ggatgttcgt
     2401 accaccaagg aattactgga gttagttgaa gcattaggtc ccaaaatttg tttactaaaa
     2461 acacatgtgg atatcttgac tgatttttcc atggagggca cagttaagcc gctaaaggca
     2521 ttatccgcca agtacaattt tttactcttc gaggacagaa aatttgctga cattggtaat
     2581 acagtcaaat tgcagtactc tgcgggtgta tacagaatag cagaatgggc agacattacg
     2641 aatgcacacg gtgtggtggg cccaggtatt gttagcggtt tgaagcaggc ggcagaagaa
     2701 gtaacaaagg aacctagagg ccttttgatg ttagcagaat tgtcatgcaa gggctcccta
     2761 tctactggag aatatactaa gggtactgtt gacattgcga agagcgacaa agattttgtt
     2821 atcggcttta ttgctcaaag agacatgggt ggaagagatg aaggttacga ttggttgatt
     2881 atgacacccg gtgtgggttt agatgacaag ggagatgcat tgggtcaaca gtatagaacc
     2941 gtggatgatg tggtttctac aggatctgac attattattg ttggaagagg actatttgca
     3001 aagggaaggg atgctaaggt agagggtgaa cgttacagaa aagcaggctg ggaagcatat
     3061 ttgagaagat gcggccagca aaactaaaaa actgtattat aagtaaatgc atgtatacta
     3121 aactcacaaa ttagagcttc aatttaatta tatcagttat tacccgagtt tcacaatgga
     3181 gggagtgaaa tctaaaagtg gaaaaaaaaa ggggaggcga aaaacaaaaa aaaaaaaaag
     3241 aacgaaccga actatgtcca caggagcaga agcagaagct gcggcagcag cttctgcggc
     3301 tttgaaagag gaaacgcgta gatgaaagaa tgatccaagc ccggctttcc ctctgtgcgc
     3361 ggattgttat ttttttagac ttatttctat gtttgttttg gacctaatat aacttagtca
     3421 tgaatcctgg ttgactcgcg cgcatctttt tccactctgc gatgattcac agtgcagcgt
     3481 gcagctcagc tccagcagca gtaccagcgg cagctccaac agtggagcct tcctttcacg
     3541 aacggaatag aaacaggaag gaaaaaaata gaccaacaag aggacccatc cgcgacttct
     3601 aatttttggc gcatgaaccg cgctgttcta cccgcccgtc cgtcctcatc cctccccccc
     3661 aggaaaacag gaaattggga aacctaggtg cgtgcatttt cgttttgggc gatgattgac
     3721 aatgagactt agtccgcttg gttgggtttc gtttcaaaaa caaggtttgg aagaagatct
     3781 tgggagagaa agaccgagag atcttgacca gggcgaagac gaccagtagc ttttggtata
     3841 gccgggataa gcttcaggca ccctagggtt gtataagagt tgtatgcaag gatgtgcgca
     3901 agggtttctg aagcaggatg gaaatgtggt gtgttttttt gtagagcaat tgccttaaag
     3961 tcggggatta cgccgagaca gcagcttgac ggttccgtgg taatatgtgt ccggcaactt
     4021 ggcgcgcctc agccgagcgg ccgcgtgtta caaccaatta accaattctg attagaaaaa
     4081 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     4141 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     4201 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     4261 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     4321 tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     4381 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     4441 gaggcgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     4501 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     4561 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     4621 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     4681 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     4741 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     4801 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctggagca
     4861 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     4921 cagttttatt gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg
     4981 agacacaacg tggctttgtt gaataaatcg aacttttgct gagttgaagg atcagtcatg
     5041 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5101 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5161 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5221 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     5281 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5341 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5401 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5461 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     5521 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5581 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5641 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5701 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     5761 ttctttcctg cgttatcccc tgattctgtg gataaccgtg cggccgcccc tttcaggcgc
     5821 g
//
