LOCUS       sequence-310205-        6453 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 6453)
  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 6453)
  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..6453
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1201..1212
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1219..1932
                     /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      1944..2015
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2031..2058
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     gene            2305..3660
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     CDS             2649..3458
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      3463..3660
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(4718..5533)
                     /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(4718..5533)
                     /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     5525..5544
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5758..6346
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6247..6266
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccctttcagg cgcgccaccg aatattaaat aatctttttt tatttcttat gtacgcctgc
       61 tttgtatgat acaaaatcat cgtatcgtat tcggcttcat gttatccgtc gaatttagat
      121 taccttggct aaggggccaa ggtggatgat tcgtatgagt atcacaattt acaaattaca
      181 agttttgcaa agagtatgta aactgtaaaa aagaaaacat tgacaaatat acctgaaaag
      241 agtccatttt ctacatttga gaaaaaaagg aataaagtga ttctcctcat cgctcataaa
      301 aaaatactat tcatgagcaa aattgagaaa aatggcaatc acattcaata ctagttagat
      361 agccatatct aaactgctgt acgtggttta atactagcac caacatcatt tgaataatgg
      421 cgaagaaacc aatttcgaaa cattcccgtg cggcaagaag atcagaagca gccgaaccgg
      481 aggctaaggc tttagagact ttacccagag ctgaaaagac agattttgca gcaacattaa
      541 tcagaactgc taacaaaaac gaagctttgc tcgaagcgaa gatccaaaag cgtggacaaa
      601 aaaataagaa gaaaaacgcg ggaagagtga gtaagcaact tatagatgac agttctttaa
      661 caagtaggaa agctattcgt gccttaaata ttaccagtag gctcgatggt aagagggaga
      721 aagccattca tagagctaaa tatgtgcagt cagctcgtaa agctggatgg gatactacaa
      781 accaaagcat taggaaagaa ttatcgacac tacaaaacgc agcaaatacg gtcagcgatt
      841 ccgttaacaa aagcacggac aaggaagggt ctgcgaatac tgaagatgcg atgatagaag
      901 acgatcaaga tcacattgaa atttacgaca gtgctaaaag ccagaaggta caagaaaaag
      961 cagtatcaac cacttttacg gcatcaaacg tatttacgtc attagaggat gagatgaatt
     1021 cgcatctaga ctgatgagac gaacgtgaga ccgaaagtga aacgtgattt catgcgtcat
     1081 tttgaacatt ttgtaaatct tatttaataa tgtgtgcggc aattcacatt taatttatga
     1141 atgttttctt aacatcgcgg caactcaaga aacggcaggt tcggatctta gctactagag
     1201 aaagaggaga aatactagat gcgtaaaggc gaagagctgt tcactggtgt cgtccctatt
     1261 ctggtggaac tggatggtga tgtcaacggt cataagtttt ccgtgcgtgg cgagggtgaa
     1321 ggtgacgcaa ctaatggtaa actgacgctg aagttcatct gtactactgg taaactgccg
     1381 gttccttggc cgactctggt aacgacgctg acttatggtg ttcagtgctt tgctcgttat
     1441 ccggaccata tgaagcagca tgacttcttc aagtccgcca tgccggaagg ctatgtgcag
     1501 gaacgcacga tttcctttaa ggatgacggc acgtacaaaa cgcgtgcgga agtgaaattt
     1561 gaaggcgata ccctggtaaa ccgcattgag ctgaaaggca ttgactttaa agaggacggc
     1621 aatatcctgg gccataagct ggaatacaat tttaacagcc acaatgttta catcaccgcc
     1681 gataaacaaa aaaatggcat taaagcgaat tttaaaattc gccacaacgt ggaggatggc
     1741 agcgtgcagc tggctgatca ctaccagcaa aacactccaa tcggtgatgg tcctgttctg
     1801 ctgccagaca atcactatct gagcacgcaa agcgttctgt ctaaagatcc gaacgagaaa
     1861 cgcgatcata tggttctgct ggagttcgta accgcagcgg gcatcacgca tggtatggat
     1921 gaactgtaca aatgaccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
     1981 ttcgttttat ctgttgtttg tcggtgaacg ctctctacta gagtcacact ggctcacctt
     2041 cgggtgggcc tttctgcgtt tataggtctc agctggaaat ctgctcgtca gtggtgctca
     2101 cactgacgaa tcatgtacag atcataccga tgactgcctg gcgactcaca actaagcaag
     2161 acagccggaa ccagcgccgg cgaacaccac tgcatatatg gcatatcaca acagtccacg
     2221 tctcaagcag ttacagagat gttacgaacc actagtgcac tgcagtacaa gcttgcctcg
     2281 tccccgccgg gtcacccggc cagcgacatg gaggcccaga ataccctcct tgacagtctt
     2341 gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt acatttagcc catacatccc
     2401 catgtataat catttgcatc catacatttt gatggccgca cggcgcgaag caaaaattac
     2461 ggctcctcgc tccagacctg cgagcaggga aacgctcccc tcacagacgc gttgaattgt
     2521 ccccacgccg cgcccctgta gagaaatata aaaggttagg atttgccact gaggttcttc
     2581 tttcatatac ttccttttaa aatcttgcta ggatacagtt ctcacatcac atccgaacat
     2641 aaacaaaaat gggtaaggaa aagactcacg tttcgaggcc gcgattaaat tccaacatgg
     2701 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
     2761 tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta
     2821 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
     2881 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
     2941 cgatccccgg caaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
     3001 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc
     3061 cttttaacag cgatcgcgta tttcgtctgg ctcaggcgca atcacgaatg aataacggtt
     3121 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
     3181 aagaaatgca taagcttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
     3241 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag
     3301 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
     3361 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
     3421 aattgcagtt tcatttgatg ctcgatgagt ttttctaaag taactgacaa taaaaagatt
     3481 cttgttttca agaacttgtc atttgtatag tttttttata ttgtagttgt tctattttaa
     3541 tcaaatgtta gcgtgattta tatttttttt cgcctcgaca tcatctgccc agatgcgaag
     3601 ttaagtgcgc agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt cgctatactg
     3661 gagttgaagc atagaagaaa tgtttaacca aaaactatcc tttttattta ttttgaattt
     3721 tctctctcat atttttgtaa aacgatactt taaccacact caagcttttg cttgctactc
     3781 aaatacctga aatctttata gacaattttc cttcttattc gtaataataa aactatattg
     3841 ttctcttcct gttttttttt ttaaaaaaaa aaactgtcaa aatctccccc acagaaaaat
     3901 aaaaacaagt tatacttttg ggagtaaacc atgaacttaa aacaacagtt catattctta
     3961 agtagtgcca ctatagttaa ccagagacaa tttaactcct aaatcttcat actgaagatt
     4021 gacataaact aaataatgac tacactgtta cttttgagtc agttgggtct tttggcattc
     4081 gccaatgcta ttaaccctat ggtaaagggc gaatatagaa ttgaacatcc taagatagga
     4141 gacttgtcag aaattgaatc tgctatctct cccaaatctt tctatgaatc atttcaatat
     4201 ggttccgaag cggagttccg taagaattgg gtttcttctg atctaacaat ggaacatgta
     4261 gacgaaacat cttcgaaggc gttccctggc acatggaagt tgcagtcacc atttttggtc
     4321 ccaggattca gtgatgatct atctttagtt ttgggtagcg aacaaactag agctgctatt
     4381 gcccataggt taaaatacga tctatcgatc aaggataatg agaaactagt ggtgcaatat
     4441 gaagttaaac ttcaaaaaat gcttgaatgt ggcggtgcat atatcaaatt gttgaaaacc
     4501 tccgcaactg atctttctca gtacgaccac tcgtcagatg actatgctgt catctttggc
     4561 ccagactttt gccaaggtta tgtggacgaa attcatttgg ttttgaagcg cgagaatcct
     4621 ttttctggaa aatctgaaga caaattctta ttaaatgccc caaaacggcg cgcctcagcc
     4681 gagcggccgc gtgttacaac caattaacca attctgatta gaaaaactca tcgagcatca
     4741 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     4801 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     4861 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     4921 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     4981 gcttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     5041 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgagg cgaaatacgc
     5101 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     5161 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     5221 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     5281 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     5341 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     5401 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     5461 catataaatc agcatccatg ttggaattta atcgcggcct ggagcaagac gtttcccgtt
     5521 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     5581 atgatgatat atttttatct tgtgcaatgt aacatcagag attttgagac acaacgtggc
     5641 tttgttgaat aaatcgaact tttgctgagt tgaaggatca gtcatgacca aaatccctta
     5701 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5761 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5821 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5881 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     5941 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6001 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     6061 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     6121 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     6181 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     6241 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6301 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6361 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     6421 atcccctgat tctgtggata accgtgcggc cgc
//
