LOCUS       sequence-317964-        6139 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting integration site I3 
            described in (Rajkumar et al.,2019). G418 selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6139)
  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 6139)
  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-G418
FEATURES             Location/Qualifiers
     source          1..6139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1096..1107
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1114..1827
                     /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      1839..1910
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1926..1953
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     gene            2204..3559
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     CDS             2548..3357
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      3362..3559
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(4450..5265)
                     /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(4450..5265)
                     /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     5257..5276
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5490..6078
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5979..5998
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tctttcctgc gttatcccct gattctgtgg ataaccgtgc ggccgcccct ttcaggcgcg
       61 cctcataccg tttatgattg catttacgtt actgaccgga gttttgtccg tatttgtgaa
      121 gccgttggag ataattgagg caccaaggtt cgaaccagac tatgctatat tctacccagg
      181 aattaattgc cagtcatgcc atgctgttaa agtaccccgt tcgaaacact gttcactgtg
      241 tcggcgatgc attccattgc aagatcatca ctgcatttgg atcaacaatt gtgttggcta
      301 cggtaactat gaatacttct atcccttcct actagcgaac tgtaccattt tatcgtatgc
      361 atccatcaga ctattttcaa tgttgagagt tctgcgagtt gtgagcaaac atttcctgtc
      421 tctctttttg ttaacaacag cgtttgcatt acttgccata atatttacat actaccagtt
      481 aaagctcgtt caagaaggaa tgaccaacaa cgagcaagat aaatggtacc tcgtacatga
      541 acatatgagg gatggaaacc ttttcaagga taacaatggc tcactgtact acagaaccag
      601 ttctggtgtg gaaggaattg atgacacatt ttactctacc aacttgtacg atcacagaaa
      661 gtacaaactc aacaatcctt cgagaataca cagcgcggaa gaactcacaa atatttatga
      721 tagaggagac ttctggaaga accttgtaga gaggataagt gtcctttgaa aatctgactt
      781 taagaattct cgtagctcca tgtacataat aaatatagac taataagatt cgcataacaa
      841 actggcttca aatcataatg gcaaagaatc acaaaattcg agatttcaat ctagctaata
      901 ttcctaaccg gaattcgcat ctagactgat gagacgaacg tgagaccgaa agtgaaacgt
      961 gatttcatgc gtcattttga acattttgta aatcttattt aataatgtgt gcggcaattc
     1021 acatttaatt tatgaatgtt ttcttaacat cgcggcaact caagaaacgg caggttcgga
     1081 tcttagctac tagagaaaga ggagaaatac tagatgcgta aaggcgaaga gctgttcact
     1141 ggtgtcgtcc ctattctggt ggaactggat ggtgatgtca acggtcataa gttttccgtg
     1201 cgtggcgagg gtgaaggtga cgcaactaat ggtaaactga cgctgaagtt catctgtact
     1261 actggtaaac tgccggttcc ttggccgact ctggtaacga cgctgactta tggtgttcag
     1321 tgctttgctc gttatccgga ccatatgaag cagcatgact tcttcaagtc cgccatgccg
     1381 gaaggctatg tgcaggaacg cacgatttcc tttaaggatg acggcacgta caaaacgcgt
     1441 gcggaagtga aatttgaagg cgataccctg gtaaaccgca ttgagctgaa aggcattgac
     1501 tttaaagagg acggcaatat cctgggccat aagctggaat acaattttaa cagccacaat
     1561 gtttacatca ccgccgataa acaaaaaaat ggcattaaag cgaattttaa aattcgccac
     1621 aacgtggagg atggcagcgt gcagctggct gatcactacc agcaaaacac tccaatcggt
     1681 gatggtcctg ttctgctgcc agacaatcac tatctgagca cgcaaagcgt tctgtctaaa
     1741 gatccgaacg agaaacgcga tcatatggtt ctgctggagt tcgtaaccgc agcgggcatc
     1801 acgcatggta tggatgaact gtacaaatga ccaggcatca aataaaacga aaggctcagt
     1861 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc
     1921 acactggctc accttcgggt gggcctttct gcgtttatag gtctcagctg gaaatctgct
     1981 cgtcagtggt gctcacactg acgaatcatg tacagatcat accgatgact gcctggcgac
     2041 tcacaactaa gcaagacagc cggaaccagc gccggcgaac accactgcat atatggcata
     2101 tcacaacagt ccacgtctca agcagttaca gagatgttac gaaccactag tgcactgcag
     2161 tacagtttag cttgcctcgt ccccgccggg tcacccggcc agcgacatgg aggcccagaa
     2221 taccctcctt gacagtcttg acgtgcgcag ctcaggggca tgatgtgact gtcgcccgta
     2281 catttagccc atacatcccc atgtataatc atttgcatcc atacattttg atggccgcac
     2341 ggcgcgaagc aaaaattacg gctcctcgct ccagacctgc gagcagggaa acgctcccct
     2401 cacagacgcg ttgaattgtc cccacgccgc gcccctgtag agaaatataa aaggttagga
     2461 tttgccactg aggttcttct ttcatatact tccttttaaa atcttgctag gatacagttc
     2521 tcacatcaca tccgaacata aacaaaaatg ggtaaggaaa agactcacgt ttcgaggccg
     2581 cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc
     2641 gggcaatcag gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt
     2701 ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac
     2761 tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat
     2821 gcatggttac tcaccactgc gatccccggc aaaacagcat tccaggtatt agaagaatat
     2881 cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg
     2941 attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctggc tcaggcgcaa
     3001 tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg
     3061 cctgttgaac aagtctggaa agaaatgcat aagcttttgc cattctcacc ggattcagtc
     3121 gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt
     3181 tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg
     3241 aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt
     3301 gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaaagt
     3361 aactgacaat aaaaagattc ttgttttcaa gaacttgtca tttgtatagt ttttttatat
     3421 tgtagttgtt ctattttaat caaatgttag cgtgatttat attttttttc gcctcgacat
     3481 catctgccca gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg
     3541 aatgctggtc gctatactgg agtaattcaa ggttgttcaa tattccgatt gttagtagat
     3601 tcagttctac cttcgctcag ttttcttaat gactcaaagg atattagaat tgaaaaattt
     3661 caaaaaagct ttaagcatag aaccacgcac ttattaaata caatggagaa atgatttggt
     3721 gggcaattac cggtttcaat tgactccgaa caggttcaaa tacaccacat ttaacagact
     3781 actaagaaag agtcaaaaag atttggctga acattaaggg aggctaggtt ttttagtgac
     3841 tttttgcgag agaaacgctt ttttagcaga ttaagcaata agtttaaaca tgtccgttga
     3901 attgtccaaa gtatccatct tgggtcagga caccattcat gtcggatacc agattcagga
     3961 acacattgtg aagactactc tggaacaatg caaatcgtcg acatatgtta tcatcaacga
     4021 tactaatgtc taccaggtgc cttactacca tgacttggtt gagctatttg agaagtctct
     4081 acccgagact tcccgtttgc tcacatatgt tgtgaagcca ggcgaggcta acaagacccg
     4141 tgaaactaag gcacagattg aagactttat gttgaatgaa gggtgcactc gtgataccgt
     4201 cgttattgca attggtggtg gtgtgatcgg tgatatgatt gggtttgttg ctgcgacatt
     4261 catgagaggt gttcgtttca ttcaaattcc tacatcgcta ttggcaatgg ttgactcgtc
     4321 tattggtggt aagactgccg ttgacacccc aatcggtaaa aacttcattg gtgccttctg
     4381 gcaaccacaa tttgtgttgg cgcgcctcag ccgagcggcc gcgtgttaca accaattaac
     4441 caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     4501 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     4561 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     4621 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     4681 agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc
     4741 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     4801 tcgtgattgc gcctgagcga ggcgaaatac gcgatcgctg ttaaaaggac aattacaaac
     4861 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     4921 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     4981 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     5041 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     5101 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     5161 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     5221 taatcgcggc ctggagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     5281 actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat
     5341 gtaacatcag agattttgag acacaacgtg gctttgttga ataaatcgaa cttttgctga
     5401 gttgaaggat cagtcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     5461 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     5521 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     5581 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     5641 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     5701 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     5761 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     5821 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     5881 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     5941 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     6001 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6061 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6121 tggccttttg ctcacatgt
//
