LOCUS       sequence-317860-        6165 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting integration site I2 
            described in (Rajkumar et al.,2019). Histidine selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6165)
  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 6165)
  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-HIS
FEATURES             Location/Qualifiers
     source          1..6165
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1230..1241
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1248..1961
                     /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      1973..2044
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2060..2087
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        2348..2535
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             2536..3198
                     /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(4459..5274)
                     /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(4459..5274)
                     /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     5182..5201
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5266..5285
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5499..6087
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5988..6007
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgattctg tggataaccg tgcggccgcc cctttcaggc gcgccaccga atattaaata
       61 atcttttttt atttcttatg tacgcctgct ttgtatgata caaaatcatc gtatcgtatt
      121 cggcttcatg ttatccgtcg aatttagatt accttggcta aggggccaag gtggatgatt
      181 cgtatgagta tcacaattta caaattacaa gttttgcaaa gagtatgtaa actgtaaaaa
      241 agaaaacatt gacaaatata cctgaaaaga gtccattttc tacatttgag aaaaaaagga
      301 ataaagtgat tctcctcatc gctcataaaa aaatactatt catgagcaaa attgagaaaa
      361 atggcaatca cattcaatac tagttagata gccatatcta aactgctgta cgtggtttaa
      421 tactagcacc aacatcattt gaataatggc gaagaaacca atttcgaaac attcccgtgc
      481 ggcaagaaga tcagaagcag ccgaaccgga ggctaaggct ttagagactt tacccagagc
      541 tgaaaagaca gattttgcag caacattaat cagaactgct aacaaaaacg aagctttgct
      601 cgaagcgaag atccaaaagc gtggacaaaa aaataagaag aaaaacgcgg gaagagtgag
      661 taagcaactt atagatgaca gttctttaac aagtaggaaa gctattcgtg ccttaaatat
      721 taccagtagg ctcgatggta agagggagaa agccattcat agagctaaat atgtgcagtc
      781 agctcgtaaa gctggatggg atactacaaa ccaaagcatt aggaaagaat tatcgacact
      841 acaaaacgca gcaaatacgg tcagcgattc cgttaacaaa agcacggaca aggaagggtc
      901 tgcgaatact gaagatgcga tgatagaaga cgatcaagat cacattgaaa tttacgacag
      961 tgctaaaagc cagaaggtac aagaaaaagc agtatcaacc acttttacgg catcaaacgt
     1021 atttacgtca ttagaggatg agatgaattc gcatctagac tgatgagacg aacgtgagac
     1081 cgaaagtgaa acgtgatttc atgcgtcatt ttgaacattt tgtaaatctt atttaataat
     1141 gtgtgcggca attcacattt aatttatgaa tgttttctta acatcgcggc aactcaagaa
     1201 acggcaggtt cggatcttag ctactagaga aagaggagaa atactagatg cgtaaaggcg
     1261 aagagctgtt cactggtgtc gtccctattc tggtggaact ggatggtgat gtcaacggtc
     1321 ataagttttc cgtgcgtggc gagggtgaag gtgacgcaac taatggtaaa ctgacgctga
     1381 agttcatctg tactactggt aaactgccgg ttccttggcc gactctggta acgacgctga
     1441 cttatggtgt tcagtgcttt gctcgttatc cggaccatat gaagcagcat gacttcttca
     1501 agtccgccat gccggaaggc tatgtgcagg aacgcacgat ttcctttaag gatgacggca
     1561 cgtacaaaac gcgtgcggaa gtgaaatttg aaggcgatac cctggtaaac cgcattgagc
     1621 tgaaaggcat tgactttaaa gaggacggca atatcctggg ccataagctg gaatacaatt
     1681 ttaacagcca caatgtttac atcaccgccg ataaacaaaa aaatggcatt aaagcgaatt
     1741 ttaaaattcg ccacaacgtg gaggatggca gcgtgcagct ggctgatcac taccagcaaa
     1801 acactccaat cggtgatggt cctgttctgc tgccagacaa tcactatctg agcacgcaaa
     1861 gcgttctgtc taaagatccg aacgagaaac gcgatcatat ggttctgctg gagttcgtaa
     1921 ccgcagcggg catcacgcat ggtatggatg aactgtacaa atgaccaggc atcaaataaa
     1981 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     2041 tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt ataggtctca
     2101 gctggaaatc tgctcgtcag tggtgctcac actgacgaat catgtacaga tcataccgat
     2161 gactgcctgg cgactcacaa ctaagcaaga cagccggaac cagcgccggc gaacaccact
     2221 gcatatatgg catatcacaa cagtccacgt ctcaagcagt tacagagatg ttacgaacca
     2281 ctagtgcact gcagtacaaa cacagtcctt tcccgcaatt ttctttttct attactcttg
     2341 gcctcctcta gtacactcta tattttttta tgcctcggta atgattttca tttttttttt
     2401 tccacctagc ggatgactct ttttttttct tagcgattgg cattatcaca taatgaatta
     2461 tacattatat aaagtaatgt gatttcttcg aagaatatac taaaaaatga gcaggcaaga
     2521 taaacgaagg caaagatgac agagcagaaa gccctagtaa agcgtattac aaatgaaacc
     2581 aagattcaga ttgcgatctc tttaaagggt ggtcccctag cgatagagca ctcgatcttc
     2641 ccagaaaaag aggcagaagc agtagcagaa caggccacac aatcgcaagt gattaacgtc
     2701 cacacaggta tagggtttct ggaccatatg atacatgctc tggccaagca ttccggctgg
     2761 tcgctaatcg ttgagtgcat tggtgactta cacatagacg accatcacac cactgaggac
     2821 tgcgggattg ctctcggtca agcttttaaa gaggccctag gggccgtgcg tggagtaaaa
     2881 aggtttggat caggatttgc gcctttggat gaggcacttt ccagagcggt ggttgatctt
     2941 tcgaacaggc cgtacgcagt tgtcgaactt ggtttgcaaa gggagaaagt aggtgatctc
     3001 tcttgcgaga tgatcccgca ttttcttgaa agctttgcag aggctagcag aattaccctc
     3061 cacgttgatt gtctgcgagg caagaatgat catcaccgta gtgagagtgc gttcaaggct
     3121 cttgcggttg ccataagaga agccacctcg cccaatggta ccaacgatgt tccctccacc
     3181 aaaggtgttc ttatgtagtg acaccgatta tttaaagctg ctgcatacga tatatataca
     3241 tgtgtatata tgtataccta tgaatgtcag taagtatgta tacgaacagt atgatactga
     3301 agatgacaag gtaatgcatc attctatacg tgtcattctg aacgaggcgc gctttccttt
     3361 tttctttttg ctttttcttt ttttttctct tgaactcgac ggatcataga gttgaagcat
     3421 agaagaaatg tttaaccaaa aactatcctt tttatttatt ttgaattttc tctctcatat
     3481 ttttgtaaaa cgatacttta accacactca agcttttgct tgctactcaa atacctgaaa
     3541 tctttataga caattttcct tcttattcgt aataataaaa ctatattgtt ctcttcctgt
     3601 tttttttttt aaaaaaaaaa actgtcaaaa tctcccccac agaaaaataa aaacaaatta
     3661 tacttttggg agtaaaccat gaacttaaaa caacagttca tattcttaag tagtgccact
     3721 atagttaacc agagacaatt taactcctaa atcttcatac tgaagattga cataaactaa
     3781 ataatgacta cactgttact tttgagtcag ttgggtcttt tggcattcgc caatgctatt
     3841 aaccctatgg taaagggcga atatagaatt gaacatccta agataggaga cttgtcagaa
     3901 attgaatctg ctatctctcc caaatctttc tatgaatcat ttcaatatgg ttccgaagcg
     3961 gagttccgta agaattgggt ttcttctgat ctaacaatgg aacatgtaga cgaaacatct
     4021 tcgaaggcgt tccctggcac atggaagttg cagtcaccat ttttggtccc aggattcagt
     4081 gatgatctat ctttagtttt gggtagcgaa caaactagag ctgctattgc ccataggtta
     4141 aaatacgatc tatcgatcaa ggataatgag aaactagtgg tgcaatatga agttaaactt
     4201 caaaaaatgc ttgaatgtgg cggtgcatat atcaaattgt tgaaaacctc cgcaactgat
     4261 ctttctcagt acgaccactc gtcagatgac tatgctgtca tctttggccc agacttttgc
     4321 caaggttatg tggacgaaat tcatttggtt ttgaagcgcg agaatccttt ttctggaaaa
     4381 tctgaagaca aattcttatt aaatgccggc gcgcctcagc cgagcggccg cgtgttacaa
     4441 ccaattaacc aattctgatt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     4501 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     4561 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     4621 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     4681 atcaccatga gtgacgactg aatccggtga gaatggcaaa agcttatgca tttctttcca
     4741 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     4801 gttattcatt cgtgattgcg cctgagcgag gcgaaatacg cgatcgctgt taaaaggaca
     4861 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     4921 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     4981 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     5041 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     5101 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     5161 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     5221 gttggaattt aatcgcggcc tggagcaaga cgtttcccgt tgaatatggc tcataacacc
     5281 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgatgata tatttttatc
     5341 ttgtgcaatg taacatcaga gattttgaga cacaacgtgg ctttgttgaa taaatcgaac
     5401 ttttgctgag ttgaaggatc agtcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5461 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5521 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5581 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5641 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5701 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5761 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5821 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5881 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5941 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6001 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6061 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6121 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcc
//
