LOCUS       sequence-318035-        6157 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting integration site I4 
            described in (Rajkumar et al.,2019). G418 selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6157)
  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 6157)
  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-G418
FEATURES             Location/Qualifiers
     source          1..6157
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1101..1112
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1119..1832
                     /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      1844..1915
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1931..1958
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     gene            2209..3564
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     CDS             2553..3362
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      3367..3564
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(4471..5286)
                     /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(4471..5286)
                     /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     5278..5297
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5511..6099
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6000..6019
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgttctttcc tgcgttatcc cctgattctg tggataaccg tgcggccgcc cctttcaggc
       61 gcgccggtgt tgcattagta tcgccgtgga tgaagatgaa ggtggaatag acgttaccca
      121 tagatacccg aatgagtatc tgattgagga tttttgactt acttacttct tcgagataca
      181 ctactctgcc gaaatttcgg actggaatgc gtcaaagtga atccactttg caggaaaacg
      241 caggaaaaac ggattcgaaa acgcaacaaa aaaaaaaaaa ttatacataa taaaattcga
      301 atcaaagcaa caagttcacg aatccattaa agcagtcaga atccaggaaa aaaaagaaac
      361 agagccagaa aaaaggcagc aacgaatcac aaccacagcg tattgcgtca gtggaaatta
      421 attaccttgg tatttccaat gctgtgtaca accaaaattt ccacatagtt agttgattcg
      481 catttccctg gtaaccgccc tcggcaacct ccctcggaaa cttccctcca gagtaacttc
      541 ccccagagtt atagagatat agagttatag tagagatata gagctataga tagattgatt
      601 cttgttgtag catttattcc gttaaacttc tgcggctaat tttgcctagc ttggcctttt
      661 ttttttgttt ttcgagcttt caaagacaaa tggacattct tacataatat attatcatat
      721 acaccgagat cggtgggtca gagcctgtag ggatggacat tttccttcta gactcgcctt
      781 ccagaccttt ttttggtttg tttctctctc cgtggaactc gctttctttt tcgccggtga
      841 tctcgaaacg taatcgatat gtattccatt ccattcgcta ttttttttgt tccataaacc
      901 atgttcttat gaacagaatt cgcatctaga ctgatgagac gaacgtgaga ccgaaagtga
      961 aacgtgattt catgcgtcat tttgaacatt ttgtaaatct tatttaataa tgtgtgcggc
     1021 aattcacatt taatttatga atgttttctt aacatcgcgg caactcaaga aacggcaggt
     1081 tcggatctta gctactagag aaagaggaga aatactagat gcgtaaaggc gaagagctgt
     1141 tcactggtgt cgtccctatt ctggtggaac tggatggtga tgtcaacggt cataagtttt
     1201 ccgtgcgtgg cgagggtgaa ggtgacgcaa ctaatggtaa actgacgctg aagttcatct
     1261 gtactactgg taaactgccg gttccttggc cgactctggt aacgacgctg acttatggtg
     1321 ttcagtgctt tgctcgttat ccggaccata tgaagcagca tgacttcttc aagtccgcca
     1381 tgccggaagg ctatgtgcag gaacgcacga tttcctttaa ggatgacggc acgtacaaaa
     1441 cgcgtgcgga agtgaaattt gaaggcgata ccctggtaaa ccgcattgag ctgaaaggca
     1501 ttgactttaa agaggacggc aatatcctgg gccataagct ggaatacaat tttaacagcc
     1561 acaatgttta catcaccgcc gataaacaaa aaaatggcat taaagcgaat tttaaaattc
     1621 gccacaacgt ggaggatggc agcgtgcagc tggctgatca ctaccagcaa aacactccaa
     1681 tcggtgatgg tcctgttctg ctgccagaca atcactatct gagcacgcaa agcgttctgt
     1741 ctaaagatcc gaacgagaaa cgcgatcata tggttctgct ggagttcgta accgcagcgg
     1801 gcatcacgca tggtatggat gaactgtaca aatgaccagg catcaaataa aacgaaaggc
     1861 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta
     1921 gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tataggtctc agctggaaat
     1981 ctgctcgtca gtggtgctca cactgacgaa tcatgtacag atcataccga tgactgcctg
     2041 gcgactcaca actaagcaag acagccggaa ccagcgccgg cgaacaccac tgcatatatg
     2101 gcatatcaca acagtccacg tctcaagcag ttacagagat gttacgaacc actagtgcac
     2161 tgcagtacag tttagcttgc ctcgtccccg ccgggtcacc cggccagcga catggaggcc
     2221 cagaataccc tccttgacag tcttgacgtg cgcagctcag gggcatgatg tgactgtcgc
     2281 ccgtacattt agcccataca tccccatgta taatcatttg catccataca ttttgatggc
     2341 cgcacggcgc gaagcaaaaa ttacggctcc tcgctccaga cctgcgagca gggaaacgct
     2401 cccctcacag acgcgttgaa ttgtccccac gccgcgcccc tgtagagaaa tataaaaggt
     2461 taggatttgc cactgaggtt cttctttcat atacttcctt ttaaaatctt gctaggatac
     2521 agttctcaca tcacatccga acataaacaa aaatgggtaa ggaaaagact cacgtttcga
     2581 ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata
     2641 atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt
     2701 tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac
     2761 taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg
     2821 atgatgcatg gttactcacc actgcgatcc ccggcaaaac agcattccag gtattagaag
     2881 aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc
     2941 attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctggctcagg
     3001 cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg
     3061 gctggcctgt tgaacaagtc tggaaagaaa tgcataagct tttgccattc tcaccggatt
     3121 cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa
     3181 taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc
     3241 tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg
     3301 gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct
     3361 aaagtaactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt atagtttttt
     3421 tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt ttttcgcctc
     3481 gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg cgtcaatcgt
     3541 atgtgaatgc tggtcgctat actggagttt cacaatggag ggagtgaaat ctaaaagtgg
     3601 aaaaaaaaag gggaggcgaa aaacaaaaaa aaaaaaaaga acgaaccgaa ctatgtccac
     3661 aggagcagaa gcagaagctg cggcagcagc ttctgcggct ttgaaagagg aaacgcgtag
     3721 atgaaagaat gatccaagcc cggctttccc tctgtgcgcg gattgttatt tttttagact
     3781 tatttctatg tttgttttgg acctaatata acttagtcat gaatcctggt tgactcgcgc
     3841 gcatcttttt ccactctgcg atgattcaca gtgcagcgtg cagctcagct ccagcagcag
     3901 taccagcggc agctccaaca gtggagcctt cctttcacga acggaataga aacaggaagg
     3961 aaaaaaatag accaacaaga ggacccatcc gcgacttcta atttttggcg catgaaccgc
     4021 gctgttctac ccgcccgtcc gtcctcatcc ctccccccca ggaaaacagg aaattgggaa
     4081 acctaggtgc gtgcattttc gttttgggcg atgattgaca atgagactta gtccgcttgg
     4141 ttgggtttcg tttcaaaaac aaggtttgga agaagatctt gggagagaaa gaccgagaga
     4201 tcttgaccag ggcgaagacg accagtagct tttggtatag ccgggataag cttcaggcac
     4261 cctagggttg tataagagtt gtatgcaagg atgtgcgcaa gggtttctga agcaggatgg
     4321 aaatgtggtg tgtttttttg tagagcaatt gccttaaagt cggggattac gccgagacag
     4381 cagcttgacg gttccgtggt aatatgtgtc cggcaacttg gcgcgcctca gccgagcggc
     4441 cgcgtgttac aaccaattaa ccaattctga ttagaaaaac tcatcgagca tcaaatgaaa
     4501 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     4561 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     4621 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     4681 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagcttatg
     4741 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     4801 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg aggcgaaata cgcgatcgct
     4861 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     4921 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     4981 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     5041 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     5101 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     5161 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     5221 atcagcatcc atgttggaat ttaatcgcgg cctggagcaa gacgtttccc gttgaatatg
     5281 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgatga
     5341 tatattttta tcttgtgcaa tgtaacatca gagattttga gacacaacgt ggctttgttg
     5401 aataaatcga acttttgctg agttgaagga tcagtcatga ccaaaatccc ttaacgtgag
     5461 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     5521 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     5581 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     5641 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     5701 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     5761 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     5821 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     5881 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     5941 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     6001 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     6061 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     6121 ttacggttcc tggccttttg ctggcctttt gctcaca
//
