LOCUS       sequence-317962-        5854 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting integration site I3 
            described in (Rajkumar et al.,2019). Histidine selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5854)
  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 5854)
  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-HIS
FEATURES             Location/Qualifiers
     source          1..5854
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1090..1101
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1108..1821
                     /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      1833..1904
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1920..1947
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        2208..2395
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             2396..3058
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     CDS             complement(4159..4974)
                     /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(4159..4974)
                     /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     4882..4901
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4966..4985
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5199..5787
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5688..5707
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctgcgttatc ccctgattct gtggataacc gtgcggccgc ccctttcagg cgcgcctcat
       61 accgtttatg attgcattta cgttactgac cggagttttg tccgtatttg tgaagccgtt
      121 ggagataatt gaggcaccaa ggttcgaacc agactatgct atattctacc caggaattaa
      181 ttgccagtca tgccatgctg ttaaagtacc ccgttcgaaa cactgttcac tgtgtcggcg
      241 atgcattcca ttgcaagatc atcactgcat ttggatcaac aattgtgttg gctacggtaa
      301 ctatgaatac ttctatccct tcctactagc gaactgtacc attttatcgt atgcatccat
      361 cagactattt tcaatgttga gagttctgcg agttgtgagc aaacatttcc tgtctctctt
      421 tttgttaaca acagcgtttg cattacttgc cataatattt acatactacc agttaaagct
      481 cgttcaagaa ggaatgacca acaacgagca agataaatgg tacctcgtac atgaacatat
      541 gagggatgga aaccttttca aggataacaa tggctcactg tactacagaa ccagttctgg
      601 tgtggaagga attgatgaca cattttactc taccaacttg tacgatcaca gaaagtacaa
      661 actcaacaat ccttcgagaa tacacagcgc ggaagaactc acaaatattt atgatagagg
      721 agacttctgg aagaaccttg tagagaggat aagtgtcctt tgaaaatctg actttaagaa
      781 ttctcgtagc tccatgtaca taataaatat agactaataa gattcgcata acaaactggc
      841 ttcaaatcat aatggcaaag aatcacaaaa ttcgagattt caatctagct aatattccta
      901 accggaattc gcatctagac tgatgagacg aacgtgagac cgaaagtgaa acgtgatttc
      961 atgcgtcatt ttgaacattt tgtaaatctt atttaataat gtgtgcggca attcacattt
     1021 aatttatgaa tgttttctta acatcgcggc aactcaagaa acggcaggtt cggatcttag
     1081 ctactagaga aagaggagaa atactagatg cgtaaaggcg aagagctgtt cactggtgtc
     1141 gtccctattc tggtggaact ggatggtgat gtcaacggtc ataagttttc cgtgcgtggc
     1201 gagggtgaag gtgacgcaac taatggtaaa ctgacgctga agttcatctg tactactggt
     1261 aaactgccgg ttccttggcc gactctggta acgacgctga cttatggtgt tcagtgcttt
     1321 gctcgttatc cggaccatat gaagcagcat gacttcttca agtccgccat gccggaaggc
     1381 tatgtgcagg aacgcacgat ttcctttaag gatgacggca cgtacaaaac gcgtgcggaa
     1441 gtgaaatttg aaggcgatac cctggtaaac cgcattgagc tgaaaggcat tgactttaaa
     1501 gaggacggca atatcctggg ccataagctg gaatacaatt ttaacagcca caatgtttac
     1561 atcaccgccg ataaacaaaa aaatggcatt aaagcgaatt ttaaaattcg ccacaacgtg
     1621 gaggatggca gcgtgcagct ggctgatcac taccagcaaa acactccaat cggtgatggt
     1681 cctgttctgc tgccagacaa tcactatctg agcacgcaaa gcgttctgtc taaagatccg
     1741 aacgagaaac gcgatcatat ggttctgctg gagttcgtaa ccgcagcggg catcacgcat
     1801 ggtatggatg aactgtacaa atgaccaggc atcaaataaa acgaaaggct cagtcgaaag
     1861 actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag agtcacactg
     1921 gctcaccttc gggtgggcct ttctgcgttt ataggtctca gctggaaatc tgctcgtcag
     1981 tggtgctcac actgacgaat catgtacaga tcataccgat gactgcctgg cgactcacaa
     2041 ctaagcaaga cagccggaac cagcgccggc gaacaccact gcatatatgg catatcacaa
     2101 cagtccacgt ctcaagcagt tacagagatg ttacgaacca ctagtgcact gcagtacaaa
     2161 cacagtcctt tcccgcaatt ttctttttct attactcttg gcctcctcta gtacactcta
     2221 tattttttta tgcctcggta atgattttca tttttttttt tccacctagc ggatgactct
     2281 ttttttttct tagcgattgg cattatcaca taatgaatta tacattatat aaagtaatgt
     2341 gatttcttcg aagaatatac taaaaaatga gcaggcaaga taaacgaagg caaagatgac
     2401 agagcagaaa gccctagtaa agcgtattac aaatgaaacc aagattcaga ttgcgatctc
     2461 tttaaagggt ggtcccctag cgatagagca ctcgatcttc ccagaaaaag aggcagaagc
     2521 agtagcagaa caggccacac aatcgcaagt gattaacgtc cacacaggta tagggtttct
     2581 ggaccatatg atacatgctc tggccaagca ttccggctgg tcgctaatcg ttgagtgcat
     2641 tggtgactta cacatagacg accatcacac cactgaggac tgcgggattg ctctcggtca
     2701 agcttttaaa gaggccctag gggccgtgcg tggagtaaaa aggtttggat caggatttgc
     2761 gcctttggat gaggcacttt ccagagcggt ggttgatctt tcgaacaggc cgtacgcagt
     2821 tgtcgaactt ggtttgcaaa gggagaaagt aggtgatctc tcttgcgaga tgatcccgca
     2881 ttttcttgaa agctttgcag aggctagcag aattaccctc cacgttgatt gtctgcgagg
     2941 caagaatgat catcaccgta gtgagagtgc gttcaaggct cttgcggttg ccataagaga
     3001 agccacctcg cccaatggta ccaacgatgt tccctccacc aaaggtgttc ttatgtagtg
     3061 acaccgatta tttaaagctg ctgcatacga tatatataca tgtgtatata tgtataccta
     3121 tgaatgtcag taagtatgta tacgaacagt atgatactga agatgacaag gtaatgcatc
     3181 attctatacg tgtcattctg aacgaggcgc gctttccttt tttctttttg ctttttcttt
     3241 ttttttctct tgaactcgac ggatcataga gtaattcaag gttgttcaat attccgattg
     3301 ttagtagatt cagttctacc ttcgctcagt tttcttaatg actcaaagga tattagaatt
     3361 gaaaaatttc aaaaaagctt taagcataga accacgcact tattaaatac aatggagaaa
     3421 tgatttggtg ggcaattacc ggtttcaatt gactccgaac aggttcaaat acaccacatt
     3481 taacagacta ctaagaaaga gtcaaaaaga tttggctgaa cattaaggga ggctaggttt
     3541 tttagtgact ttttgcgaga gaaacgcttt tttagcagat taagcaataa gtttaaacat
     3601 gtccgttgaa ttgtccaaag tatccatctt gggtcaggac accattcatg tcggatacca
     3661 gattcaggaa cacattgtga agactactct ggaacaatgc aaatcgtcga catatgttat
     3721 catcaacgat actaatgtct accaggtgcc ttactaccat gacttggttg agctatttga
     3781 gaagtctcta cccgagactt cccgtttgct cacatatgtt gtgaagccag gcgaggctaa
     3841 caagacccgt gaaactaagg cacagattga agactttatg ttgaatgaag ggtgcactcg
     3901 tgataccgtc gttattgcaa ttggtggtgg tgtgatcggt gatatgattg ggtttgttgc
     3961 tgcgacattc atgagaggtg ttcgtttcat tcaaattcct acatcgctat tggcaatggt
     4021 tgactcgtct attggtggta agactgccgt tgacacccca atcggtaaaa acttcattgg
     4081 tgccttctgg caaccacaat ttgtgttggc gcgcctcagc cgagcggccg cgtgttacaa
     4141 ccaattaacc aattctgatt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     4201 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     4261 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     4321 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     4381 atcaccatga gtgacgactg aatccggtga gaatggcaaa agcttatgca tttctttcca
     4441 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     4501 gttattcatt cgtgattgcg cctgagcgag gcgaaatacg cgatcgctgt taaaaggaca
     4561 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     4621 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     4681 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     4741 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     4801 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     4861 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     4921 gttggaattt aatcgcggcc tggagcaaga cgtttcccgt tgaatatggc tcataacacc
     4981 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgatgata tatttttatc
     5041 ttgtgcaatg taacatcaga gattttgaga cacaacgtgg ctttgttgaa taaatcgaac
     5101 ttttgctgag ttgaaggatc agtcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5161 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5221 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5281 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5341 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5401 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5461 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5521 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5581 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5641 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5701 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5761 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5821 gccttttgct ggccttttgc tcacatgttc tttc
//
