LOCUS       sequence-326333-        5872 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting integration site I4 
            described in (Rajkumar et al.,2019). Histidine selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5872)
  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 5872)
  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-HIS
FEATURES             Location/Qualifiers
     source          1..5872
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1029..1040
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1047..1760
                     /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      1772..1843
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1859..1886
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        2147..2334
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             2335..2997
                     /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(4114..4929)
                     /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(4114..4929)
                     /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     4837..4856
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4921..4940
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5154..5742
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5643..5662
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cattagtatc gccgtggatg aagatgaagg tggaatagac gttacccata gatacccgaa
       61 tgagtatctg attgaggatt tttgacttac ttacttcttc gagatacact actctgccga
      121 aatttcggac tggaatgcgt caaagtgaat ccactttgca ggaaaacgca ggaaaaacgg
      181 attcgaaaac gcaacaaaaa aaaaaaaatt atacataata aaatttgaat caaagcaaca
      241 agttcacgaa tccattaaag cagtcagaat ccaggaaaaa aaagaaacag agccagaaaa
      301 aaggcagcaa cgaatcacaa ccacagcgta ttgcgtcagt ggaaattaat taccttggta
      361 tttccaatgc tgtgtacaac caaaatttcc acatagttag ttgattcgca tttccctggt
      421 aaccgccctc ggcaacctcc ctcggaaact tccctccaga gtaacttccc ccagagttat
      481 agagatatag agttatagta gagatataga gctatagata gattgattct tgttgtagca
      541 tttattccgt taaacttctg cggctaattt tgcctagctt ggcctttttt ttttgttttt
      601 cgagctttca aagacaaatg gacattctta cataatatat tatcatatac accgagatcg
      661 gtgggtcaga gcctgtaggg atggacattt tccttctaga ctcgccttcc agaccttttt
      721 ttggtttgtt tctctctccg tggaactcgc tttctttttc gccggtgatc tcgaaacgta
      781 atcgatatgt attccattcc attcgctatt ttttttgttc cataaaccat gttcttatga
      841 acagaattcg catctagact gatgagacga acgtgagacc gaaagtgaaa cgtgatttca
      901 tgcgtcattt tgaacatttt gtaaatctta tttaataatg tgtgcggcaa ttcacattta
      961 atttatgaat gttttcttaa catcgcggca actcaagaaa cggcaggttc ggatcttagc
     1021 tactagagaa agaggagaaa tactagatgc gtaaaggcga agagctgttc actggtgtcg
     1081 tccctattct ggtggaactg gatggtgatg tcaacggtca taagttttcc gtgcgtggcg
     1141 agggtgaagg tgacgcaact aatggtaaac tgacgctgaa gttcatctgt actactggta
     1201 aactgccggt tccttggccg actctggtaa cgacgctgac ttatggtgtt cagtgctttg
     1261 ctcgttatcc ggaccatatg aagcagcatg acttcttcaa gtccgccatg ccggaaggct
     1321 atgtgcagga acgcacgatt tcctttaagg atgacggcac gtacaaaacg cgtgcggaag
     1381 tgaaatttga aggcgatacc ctggtaaacc gcattgagct gaaaggcatt gactttaaag
     1441 aggacggcaa tatcctgggc cataagctgg aatacaattt taacagccac aatgtttaca
     1501 tcaccgccga taaacaaaaa aatggcatta aagcgaattt taaaattcgc cacaacgtgg
     1561 aggatggcag cgtgcagctg gctgatcact accagcaaaa cactccaatc ggtgatggtc
     1621 ctgttctgct gccagacaat cactatctga gcacgcaaag cgttctgtct aaagatccga
     1681 acgagaaacg cgatcatatg gttctgctgg agttcgtaac cgcagcgggc atcacgcatg
     1741 gtatggatga actgtacaaa tgaccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     1801 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg
     1861 ctcaccttcg ggtgggcctt tctgcgttta taggtctcag ctggaaatct gctcgtcagt
     1921 ggtgctcaca ctgacgaatc atgtacagat cataccgatg actgcctggc gactcacaac
     1981 taagcaagac agccggaacc agcgccggcg aacaccactg catatatggc atatcacaac
     2041 agtccacgtc tcaagcagtt acagagatgt tacgaaccac tagtgcactg cagtacaaac
     2101 acagtccttt cccgcaattt tctttttcta ttactcttgg cctcctctag tacactctat
     2161 atttttttat gcctcggtaa tgattttcat tttttttttt ccacctagcg gatgactctt
     2221 tttttttctt agcgattggc attatcacat aatgaattat acattatata aagtaatgtg
     2281 atttcttcga agaatatact aaaaaatgag caggcaagat aaacgaaggc aaagatgaca
     2341 gagcagaaag ccctagtaaa gcgtattaca aatgaaacca agattcagat tgcgatctct
     2401 ttaaagggtg gtcccctagc gatagagcac tcgatcttcc cagaaaaaga ggcagaagca
     2461 gtagcagaac aggccacaca atcgcaagtg attaacgtcc acacaggtat agggtttctg
     2521 gaccatatga tacatgctct ggccaagcat tccggctggt cgctaatcgt tgagtgcatt
     2581 ggtgacttac acatagacga ccatcacacc actgaggact gcgggattgc tctcggtcaa
     2641 gcttttaaag aggccctagg ggccgtgcgt ggagtaaaaa ggtttggatc aggatttgcg
     2701 cctttggatg aggcactttc cagagcggtg gttgatcttt cgaacaggcc gtacgcagtt
     2761 gtcgaacttg gtttgcaaag ggagaaagta ggtgatctct cttgcgagat gatcccgcat
     2821 tttcttgaaa gctttgcaga ggctagcaga attaccctcc acgttgattg tctgcgaggc
     2881 aagaatgatc atcaccgtag tgagagtgcg ttcaaggctc ttgcggttgc cataagagaa
     2941 gccacctcgc ccaatggtac caacgatgtt ccctccacca aaggtgttct tatgtagtga
     3001 caccgattat ttaaagctgc tgcatacgat atatatacat gtgtatatat gtatacctat
     3061 gaatgtcagt aagtatgtat acgaacagta tgatactgaa gatgacaagg taatgcatca
     3121 ttctatacgt gtcattctga acgaggcgcg ctttcctttt ttctttttgc tttttctttt
     3181 tttttctctt gaactcgacg gatcatagag tttcacaatg gagggagtga aatctaaaag
     3241 tggaaaaaaa aaggggaggc gaaaaacaaa aaaaaaaaaa agaacgaacc gaactatgtc
     3301 cacaggagca gaagcagaag ctgcggcagc agcttctgcg gctttgaaag aggaaacgcg
     3361 tagatgaaag aatgatccaa gcccggcttt ccctctgtgc gcggattgtt atttttttag
     3421 acttatttct atgtttgttt tggacctaat ataacttagt catgaatcct ggttgactcg
     3481 cgcgcatctt tttccactct gcgatgattc acagtgcagc gtgcagctca gctccagcag
     3541 cagtaccagc ggcagctcca acagtggagc cttcctttca cgaacggaat agaaacagga
     3601 aggaaaaaaa tagaccaaca agaggaccca tccgcgactt ctaatttttg gcgcatgaac
     3661 cgcgctgttc tacccgcccg tccgtcctca tccctccccc ccaggaaaac aggaaattgg
     3721 gaaacctagg tgcgtgcatt ttcgttttgg gcgatgattg acaatgagac ttagtccgct
     3781 tggttgggtt tcgtttcaaa aacaaggttt ggaagaagat cttgggagag aaagaccgag
     3841 agatcttgac cagggcgaag acgaccagta gcttttggta tagccgggat aagcttcagg
     3901 caccctaggg ttgtataaga gttgtatgca aggatgtgcg caagggtttc tgaagcagga
     3961 tggaaatgtg gtgtgttttt ttgtagagca attgccttaa agtcggggat tacgccgaga
     4021 cagcagcttg acggttccgt ggtaatatgt gtccggcaac ttggcgcgcc tcagccgagc
     4081 ggccgcgtgt tacaaccaat taaccaattc tgattagaaa aactcatcga gcatcaaatg
     4141 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     4201 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     4261 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     4321 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt
     4381 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     4441 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgaggcgaa atacgcgatc
     4501 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     4561 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     4621 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     4681 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     4741 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     4801 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     4861 taaatcagca tccatgttgg aatttaatcg cggcctggag caagacgttt cccgttgaat
     4921 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     4981 tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacaa cgtggctttg
     5041 ttgaataaat cgaacttttg ctgagttgaa ggatcagtca tgaccaaaat cccttaacgt
     5101 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5161 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5221 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     5281 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     5341 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     5401 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     5461 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     5521 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     5581 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     5641 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     5701 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     5761 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     5821 cctgattctg tggataaccg tgcggccgcc cctttcaggc gcgccggtgt tg
//
