LOCUS       sequence-317862-        6450 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting integration site I2 
            described in (Rajkumar et al.,2019). G418 selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6450)
  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 6450)
  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-G418
FEATURES             Location/Qualifiers
     source          1..6450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1193..1204
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1211..1924
                     /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      1936..2007
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2023..2050
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     gene            2301..3656
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     CDS             2645..3454
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      3459..3656
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(4707..5522)
                     /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(4707..5522)
                     /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     5514..5533
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5747..6335
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6236..6255
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggcgcgccac cgaatattaa ataatctttt tttatttctt atgtacgcct gctttgtatg
       61 atacaaaatc atcgtatcgt attcggcttc atgttatccg tcgaatttag attaccttgg
      121 ctaaggggcc aaggtggatg attcgtatga gtatcacaat ttacaaatta caagttttgc
      181 aaagagtatg taaactgtaa aaaagaaaac attgacaaat atacctgaaa agagtccatt
      241 ttctacattt gagaaaaaaa ggaataaagt gattctcctc atcgctcata aaaaaatact
      301 attcatgagc aaaattgaga aaaatggcaa tcacattcaa tactagttag atagccatat
      361 ctaaactgct gtacgtggtt taatactagc accaacatca tttgaataat ggcgaagaaa
      421 ccaatttcga aacattcccg tgcggcaaga agatcagaag cagccgaacc ggaggctaag
      481 gctttagaga ctttacccag agctgaaaag acagattttg cagcaacatt aatcagaact
      541 gctaacaaaa acgaagcttt gctcgaagcg aagatccaaa agcgtggaca aaaaaataag
      601 aagaaaaacg cgggaagagt gagtaagcaa cttatagatg acagttcttt aacaagtagg
      661 aaagctattc gtgccttaaa tattaccagt aggctcgatg gtaagaggga gaaagccatt
      721 catagagcta aatatgtgca gtcagctcgt aaagctggat gggatactac aaaccaaagc
      781 attaggaaag aattatcgac actacaaaac gcagcaaata cggtcagcga ttccgttaac
      841 aaaagcacgg acaaggaagg gtctgcgaat actgaagatg cgatgataga agacgatcaa
      901 gatcacattg aaatttacga cagtgctaaa agccagaagg tacaagaaaa agcagtatca
      961 accactttta cggcatcaaa cgtatttacg tcattagagg atgagatgaa ttcgcatcta
     1021 gactgatgag acgaacgtga gaccgaaagt gaaacgtgat ttcatgcgtc attttgaaca
     1081 ttttgtaaat cttatttaat aatgtgtgcg gcaattcaca tttaatttat gaatgttttc
     1141 ttaacatcgc ggcaactcaa gaaacggcag gttcggatct tagctactag agaaagagga
     1201 gaaatactag atgcgtaaag gcgaagagct gttcactggt gtcgtcccta ttctggtgga
     1261 actggatggt gatgtcaacg gtcataagtt ttccgtgcgt ggcgagggtg aaggtgacgc
     1321 aactaatggt aaactgacgc tgaagttcat ctgtactact ggtaaactgc cggttccttg
     1381 gccgactctg gtaacgacgc tgacttatgg tgttcagtgc tttgctcgtt atccggacca
     1441 tatgaagcag catgacttct tcaagtccgc catgccggaa ggctatgtgc aggaacgcac
     1501 gatttccttt aaggatgacg gcacgtacaa aacgcgtgcg gaagtgaaat ttgaaggcga
     1561 taccctggta aaccgcattg agctgaaagg cattgacttt aaagaggacg gcaatatcct
     1621 gggccataag ctggaataca attttaacag ccacaatgtt tacatcaccg ccgataaaca
     1681 aaaaaatggc attaaagcga attttaaaat tcgccacaac gtggaggatg gcagcgtgca
     1741 gctggctgat cactaccagc aaaacactcc aatcggtgat ggtcctgttc tgctgccaga
     1801 caatcactat ctgagcacgc aaagcgttct gtctaaagat ccgaacgaga aacgcgatca
     1861 tatggttctg ctggagttcg taaccgcagc gggcatcacg catggtatgg atgaactgta
     1921 caaatgacca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     1981 atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg
     2041 cctttctgcg tttataggtc tcagctggaa atctgctcgt cagtggtgct cacactgacg
     2101 aatcatgtac agatcatacc gatgactgcc tggcgactca caactaagca agacagccgg
     2161 aaccagcgcc ggcgaacacc actgcatata tggcatatca caacagtcca cgtctcaagc
     2221 agttacagag atgttacgaa ccactagtgc actgcagtac agtttagctt gcctcgtccc
     2281 cgccgggtca cccggccagc gacatggagg cccagaatac cctccttgac agtcttgacg
     2341 tgcgcagctc aggggcatga tgtgactgtc gcccgtacat ttagcccata catccccatg
     2401 tataatcatt tgcatccata cattttgatg gccgcacggc gcgaagcaaa aattacggct
     2461 cctcgctcca gacctgcgag cagggaaacg ctcccctcac agacgcgttg aattgtcccc
     2521 acgccgcgcc cctgtagaga aatataaaag gttaggattt gccactgagg ttcttctttc
     2581 atatacttcc ttttaaaatc ttgctaggat acagttctca catcacatcc gaacataaac
     2641 aaaaatgggt aaggaaaaga ctcacgtttc gaggccgcga ttaaattcca acatggatgc
     2701 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     2761 tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     2821 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     2881 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     2941 ccccggcaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     3001 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     3061 taacagcgat cgcgtatttc gtctggctca ggcgcaatca cgaatgaata acggtttggt
     3121 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     3181 aatgcataag cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     3241 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     3301 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     3361 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     3421 gcagtttcat ttgatgctcg atgagttttt ctaaagtaac tgacaataaa aagattcttg
     3481 ttttcaagaa cttgtcattt gtatagtttt tttatattgt agttgttcta ttttaatcaa
     3541 atgttagcgt gatttatatt ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa
     3601 gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat gctggtcgct atactggagt
     3661 tgaagcatag aagaaatgtt taaccaaaaa ctatcctttt tatttatttt gaattttctc
     3721 tctcatattt ttgtaaaacg atactttaac cacactcaag cttttgcttg ctactcaaat
     3781 acctgaaatc tttatagaca attttccttc ttattcgtaa taataaaact atattgttct
     3841 cttcctgttt ttttttttaa aaaaaaaaac tgtcaaaatc tcccccacag aaaaataaaa
     3901 acaaattata cttttgggag taaaccatga acttaaaaca acagttcata ttcttaagta
     3961 gtgccactat agttaaccag agacaattta actcctaaat cttcatactg aagattgaca
     4021 taaactaaat aatgactaca ctgttacttt tgagtcagtt gggtcttttg gcattcgcca
     4081 atgctattaa ccctatggta aagggcgaat atagaattga acatcctaag ataggagact
     4141 tgtcagaaat tgaatctgct atctctccca aatctttcta tgaatcattt caatatggtt
     4201 ccgaagcgga gttccgtaag aattgggttt cttctgatct aacaatggaa catgtagacg
     4261 aaacatcttc gaaggcgttc cctggcacat ggaagttgca gtcaccattt ttggtcccag
     4321 gattcagtga tgatctatct ttagttttgg gtagcgaaca aactagagct gctattgccc
     4381 ataggttaaa atacgatcta tcgatcaagg ataatgagaa actagtggtg caatatgaag
     4441 ttaaacttca aaaaatgctt gaatgtggcg gtgcatatat caaattgttg aaaacctccg
     4501 caactgatct ttctcagtac gaccactcgt cagatgacta tgctgtcatc tttggcccag
     4561 acttttgcca aggttatgtg gacgaaattc atttggtttt gaagcgcgag aatccttttt
     4621 ctggaaaatc tgaagacaaa ttcttattaa atgccggcgc gcctcagccg agcggccgcg
     4681 tgttacaacc aattaaccaa ttctgattag aaaaactcat cgagcatcaa atgaaactgc
     4741 aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa
     4801 ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt
     4861 ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca
     4921 agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag cttatgcatt
     4981 tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca
     5041 accaaaccgt tattcattcg tgattgcgcc tgagcgaggc gaaatacgcg atcgctgtta
     5101 aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca
     5161 acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg
     5221 atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga
     5281 agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca
     5341 acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga
     5401 tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca
     5461 gcatccatgt tggaatttaa tcgcggcctg gagcaagacg tttcccgttg aatatggctc
     5521 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgatgatata
     5581 tttttatctt gtgcaatgta acatcagaga ttttgagaca caacgtggct ttgttgaata
     5641 aatcgaactt ttgctgagtt gaaggatcag tcatgaccaa aatcccttaa cgtgagtttt
     5701 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     5761 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     5821 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     5881 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     5941 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6001 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6061 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6121 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6181 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6241 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     6301 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     6361 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     6421 ctgtggataa ccgtgcggcc gcccctttca
//
