LOCUS       sequence-310222-        5824 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 5824)
  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 5824)
  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..5824
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1052..1063
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1070..1783
                     /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      1795..1866
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1882..1909
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             2298..3101
                     /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(4089..4904)
                     /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(4089..4904)
                     /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     4812..4831
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4896..4915
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5129..5717
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5618..5637
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccctttcagg cgcgccggtg ttgcattagt atcgccgtgg atgaagatga aggtggaata
       61 gacgttaccc atagataccc gaatgagtat ctgattgagg atttttgact tacttacttc
      121 ttcgagatac actactctgc cgaaatttcg gactggaatg cgtcaaagtg aatccacttt
      181 gcaggaaaac gcaggaaaaa cggattcgaa aacgcaacaa aaaaaaaaaa attatacata
      241 ataaaatttg aatcaaagca acaagttcac gaatccatta aagcagtcag aatccaggaa
      301 aaaaaagaaa cagagccaga aaaaaggcag caacgaatca caaccacagc gtattgcgtc
      361 agtggaaatt aattaccttg gtatttccaa tgctgtgtac aaccaaaatt tccacatagt
      421 tagttgattc gcatttccct ggtaaccgcc ctcggcaacc tccctcggaa acttccctcc
      481 agagtaactt cccccagagt tatagagata tagagttata gtagagatat agagctatag
      541 atagattgat tcttgttgta gcatttattc cgttaaactt ctgcggctaa ttttgcctag
      601 cttggccttt tttttttgtt tttcgagctt tcaaagacaa atggacattc ttacataata
      661 tattatcata tacaccgaga tcggtgggtc agagcctgta gggatggaca ttttccttct
      721 agactcgcct tccagacctt tttttggttt gtttctctct ccgtggaact cgctttcttt
      781 ttcgccggtg atctcgaaac gtaatcgata tgtattccat tccattcgct attttttttg
      841 ttccataaac catgttctta tgaacagaat tcgcatctag actgatgaga cgaacgtgag
      901 accgaaagtg aaacgtgatt tcatgcgtca ttttgaacat tttgtaaatc ttatttaata
      961 atgtgtgcgg caattcacat ttaatttatg aatgttttct taacatcgcg gcaactcaag
     1021 aaacggcagg ttcggatctt agctactaga gaaagaggag aaatactaga tgcgtaaagg
     1081 cgaagagctg ttcactggtg tcgtccctat tctggtggaa ctggatggtg atgtcaacgg
     1141 tcataagttt tccgtgcgtg gcgagggtga aggtgacgca actaatggta aactgacgct
     1201 gaagttcatc tgtactactg gtaaactgcc ggttccttgg ccgactctgg taacgacgct
     1261 gacttatggt gttcagtgct ttgctcgtta tccggaccat atgaagcagc atgacttctt
     1321 caagtccgcc atgccggaag gctatgtgca ggaacgcacg atttccttta aggatgacgg
     1381 cacgtacaaa acgcgtgcgg aagtgaaatt tgaaggcgat accctggtaa accgcattga
     1441 gctgaaaggc attgacttta aagaggacgg caatatcctg ggccataagc tggaatacaa
     1501 ttttaacagc cacaatgttt acatcaccgc cgataaacaa aaaaatggca ttaaagcgaa
     1561 ttttaaaatt cgccacaacg tggaggatgg cagcgtgcag ctggctgatc actaccagca
     1621 aaacactcca atcggtgatg gtcctgttct gctgccagac aatcactatc tgagcacgca
     1681 aagcgttctg tctaaagatc cgaacgagaa acgcgatcat atggttctgc tggagttcgt
     1741 aaccgcagcg ggcatcacgc atggtatgga tgaactgtac aaatgaccag gcatcaaata
     1801 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     1861 gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttataggtct
     1921 cagctggaaa tctgctcgtc agtggtgctc acactgacga atcatgtaca gatcataccg
     1981 atgactgcct ggcgactcac aactaagcaa gacagccgga accagcgccg gcgaacacca
     2041 ctgcatatat ggcatatcac aacagtccac gtctcaagca gttacagaga tgttacgaac
     2101 cactagtgca ctgcagtaca cggtttcctt gaaatttttt tgattcggta atctccgaac
     2161 agaaggaaga acgaaggaag gagcacagac ttagattggt atatatacgc atatgtagtg
     2221 ttgaagaaac atgaaattgc ccagtattct taacccaact gcacagaaca aaaacgtgca
     2281 ggaaacgaag ataaatcatg tcgaaagcta catataagga acgtgctgct actcatccta
     2341 gtcctgttgc tgccaagcta tttaatatca tgcacgaaaa gcaaacaaac ttgtgtgctt
     2401 cattggatgt tcgtaccacc aaggaattac tggagttagt tgaagcatta ggtcccaaaa
     2461 tttgtttact aaaaacacat gtggatatct tgactgattt ttccatggag ggcacagtta
     2521 agccgctaaa ggcattatcc gccaagtaca attttttact cttcgaggac agaaaatttg
     2581 ctgacattgg taatacagtc aaattgcagt actctgcggg tgtatacaga atagcagaat
     2641 gggcagacat tacgaatgca cacggtgtgg tgggcccagg tattgttagc ggtttgaagc
     2701 aggcggcaga agaagtaaca aaggaaccta gaggcctttt gatgttagca gaattgtcat
     2761 gcaagggctc cctatctact ggagaatata ctaagggtac tgttgacatt gcgaagagcg
     2821 acaaagattt tgttatcggc tttattgctc aaagagacat gggtggaaga gatgaaggtt
     2881 acgattggtt gattatgaca cccggtgtgg gtttagatga caagggagat gcattgggtc
     2941 aacagtatag aaccgtggat gatgtggttt ctacaggatc tgacattatt attgttggaa
     3001 gaggactatt tgcaaaggga agggatgcta aggtagaggg tgaacgttac agaaaagcag
     3061 gctgggaagc atatttgaga agatgcggcc agcaaaacta aaaaactgta ttataagtaa
     3121 atgcatgtat actaaactca caaattagag cttcaattta attatatcag ttattacccg
     3181 agtttcacaa tggagggagt gaaatctaaa agtggaaaaa aaaaggggag gcgaaaaaca
     3241 aaaaaaaaaa aaagaacgaa ccgaactatg tccacaggag cagaagcaga agctgcggca
     3301 gcagcttctg cggctttgaa agaggaaacg cgtagatgaa agaatgatcc aagcccggct
     3361 ttccctctgt gcgcggattg ttattttttt agacttattt ctatgtttgt tttggaccta
     3421 atataactta gtcatgaatc ctggttgact cgcgcgcatc tttttccact ctgcgatgat
     3481 tcacagtgca gcgtgcagct cagctccagc agcagtacca gcggcagctc caacagtgga
     3541 gccttccttt cacgaacgga atagaaacag gaaggaaaaa aatagaccaa caagaggacc
     3601 catccgcgac ttctaatttt tggcgcatga accgcgctgt tctacccgcc cgtccgtcct
     3661 catccctccc ccccaggaaa acaggaaatt gggaaaccta ggtgcgtgca ttttcgtttt
     3721 gggcgatgat tgacaatgag acttagtccg cttggttggg tttcgtttca aaaacaaggt
     3781 ttggaagaag atcttgggag agaaagaccg agagatcttg accagggcga agacgaccag
     3841 tagcttttgg tatagccggg ataagcttca ggcaccctag ggttgtataa gagttgtatg
     3901 caaggatgtg cgcaagggtt tctgaagcag gatggaaatg tggtgtgttt ttttgtagag
     3961 caattgcctt aaagtcgggg attacgccga gacagcagct tgacggttcc gtggtaatat
     4021 gtgtccggca acaggcgcgc ctcagccgac cgagcggccg cgtgttacaa ccaattaacc
     4081 aattctgatt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     4141 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     4201 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     4261 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     4321 gtgacgactg aatccggtga gaatggcaaa agcttatgca tttctttcca gacttgttca
     4381 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     4441 cgtgattgcg cctgagcgag gcgaaatacg cgatcgctgt taaaaggaca attacaaaca
     4501 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     4561 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     4621 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     4681 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     4741 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     4801 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     4861 aatcgcggcc tggagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta
     4921 ctgtttatgt aagcagacag ttttattgtt catgatgata tatttttatc ttgtgcaatg
     4981 taacatcaga gattttgaga cacaacgtgg ctttgttgaa taaatcgaac ttttgctgag
     5041 ttgaaggatc agtcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5101 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5161 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5221 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     5281 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5341 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5401 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5461 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5521 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5581 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5641 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5701 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5761 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtgcgg
     5821 ccgc
//
