LOCUS       sequence-317075-        6213 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting the LAC4 locus. G418 
            selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6213)
  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 6213)
  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: pI1-MTU-DO-G418
FEATURES             Location/Qualifiers
     source          1..6213
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1146..1157
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1164..1877
                     /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      1889..1960
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1976..2003
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     gene            2254..3609
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     CDS             2598..3407
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      3412..3609
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(4544..5359)
                     /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(4544..5359)
                     /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     5351..5370
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5584..6172
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6073..6092
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtgc
       61 ggccgcccct ttcaggcgcg ccgaaagtga ttgaagaacc ctcaaacttg ctggatattt
      121 ccttaccctt gacctttagg ctttcaattt tacccaacaa tttgtccaag acaaattgca
      181 atccactgga ttcaactgag acataacgtt taccgtcgtt gatcttggta ggtttttctg
      241 ctgtctctgt aacaaaatct ggtaccttta atggaagttc ggcttggccc caggcaattt
      301 catgacctgc ctttagaaca ccagcatcat cctttaacac ggcaacaaca taagtggtat
      361 cagaaggaat agtaacagat tcttctggct ttaaagctgg aacgtcgatt gtctttcccg
      421 tgtccttgtc gataaacaat aagtgatctg tcgtaatgaa gtcgtgctta tttgtgattg
      481 ttacagatcc gtgcgcaatt ttaatatgaa cgggttcaat aaccttctta tactctacaa
      541 ggcccggagt aggattatgc tcactgttac acaaaccatc catgatgaac actccgtcat
      601 gaacctcttc cttaaagtca ccaccataag cataagcttt atgcaactta ccatctgcag
      661 tactaacatc ttcgaattca ataccgtgat ttgcccattc ccagataaag ccaccttggt
      721 aaaacttctc cttgtagaac aactcttgat attctttcaa agaacccggg ccgttaccca
      781 ttgcatgacc gtactcacac aagatcaaag gcttttcaaa cttaccattt tcatcagtgt
      841 ggttcttcct ccacctttcc ataatttcaa atgttgggta catgaaacta aagatatctg
      901 cactcaaagc gttcaagtca ccctcataat gcaccagtct ggtaggatcc aattgcttaa
      961 gaattcgcat ctagactgat gagacgaacg tgagaccgaa agtgaaacgt gatttcatgc
     1021 gtcattttga acattttgta aatcttattt aataatgtgt gcggcaattc acatttaatt
     1081 tatgaatgtt ttcttaacat cgcggcaact caagaaacgg caggttcgga tcttagctac
     1141 tagagaaaga ggagaaatac tagatgcgta aaggcgaaga gctgttcact ggtgtcgtcc
     1201 ctattctggt ggaactggat ggtgatgtca acggtcataa gttttccgtg cgtggcgagg
     1261 gtgaaggtga cgcaactaat ggtaaactga cgctgaagtt catctgtact actggtaaac
     1321 tgccggttcc ttggccgact ctggtaacga cgctgactta tggtgttcag tgctttgctc
     1381 gttatccgga ccatatgaag cagcatgact tcttcaagtc cgccatgccg gaaggctatg
     1441 tgcaggaacg cacgatttcc tttaaggatg acggcacgta caaaacgcgt gcggaagtga
     1501 aatttgaagg cgataccctg gtaaaccgca ttgagctgaa aggcattgac tttaaagagg
     1561 acggcaatat cctgggccat aagctggaat acaattttaa cagccacaat gtttacatca
     1621 ccgccgataa acaaaaaaat ggcattaaag cgaattttaa aattcgccac aacgtggagg
     1681 atggcagcgt gcagctggct gatcactacc agcaaaacac tccaatcggt gatggtcctg
     1741 ttctgctgcc agacaatcac tatctgagca cgcaaagcgt tctgtctaaa gatccgaacg
     1801 agaaacgcga tcatatggtt ctgctggagt tcgtaaccgc agcgggcatc acgcatggta
     1861 tggatgaact gtacaaatga ccaggcatca aataaaacga aaggctcagt cgaaagactg
     1921 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc acactggctc
     1981 accttcgggt gggcctttct gcgtttatag gtctcagctg gaaatctgct cgtcagtggt
     2041 gctcacactg acgaatcatg tacagatcat accgatgact gcctggcgac tcacaactaa
     2101 gcaagacagc cggaaccagc gccggcgaac accactgcat atatggcata tcacaacagt
     2161 ccacgtctca agcagttaca gagatgttac gaaccactag tgcactgcag tacagtttag
     2221 cttgcctcgt ccccgccggg tcacccggcc agcgacatgg aggcccagaa taccctcctt
     2281 gacagtcttg acgtgcgcag ctcaggggca tgatgtgact gtcgcccgta catttagccc
     2341 atacatcccc atgtataatc atttgcatcc atacattttg atggccgcac ggcgcgaagc
     2401 aaaaattacg gctcctcgct ccagacctgc gagcagggaa acgctcccct cacagacgcg
     2461 ttgaattgtc cccacgccgc gcccctgtag agaaatataa aaggttagga tttgccactg
     2521 aggttcttct ttcatatact tccttttaaa atcttgctag gatacagttc tcacatcaca
     2581 tccgaacata aacaaaaatg ggtaaggaaa agactcacgt ttcgaggccg cgattaaatt
     2641 ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag
     2701 gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg
     2761 gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg
     2821 aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac
     2881 tcaccactgc gatccccggc aaaacagcat tccaggtatt agaagaatat cctgattcag
     2941 gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt
     3001 gtaattgtcc ttttaacagc gatcgcgtat ttcgtctggc tcaggcgcaa tcacgaatga
     3061 ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac
     3121 aagtctggaa agaaatgcat aagcttttgc cattctcacc ggattcagtc gtcactcatg
     3181 gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg
     3241 ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg
     3301 gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg
     3361 atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaaagt aactgacaat
     3421 aaaaagattc ttgttttcaa gaacttgtca tttgtatagt ttttttatat tgtagttgtt
     3481 ctattttaat caaatgttag cgtgatttat attttttttc gcctcgacat catctgccca
     3541 gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg aatgctggtc
     3601 gctatactgg agttgcttaa ttagcttgta catggctttg tggtttctgc cataacaagc
     3661 ttcgttaccc aaggaccaga taataatcga aggatgattg acatctctta ggacaagttg
     3721 ggaagctctg tctaagtacg cgacctcgta ctctggatta tctgataagt aatgggcatt
     3781 aacatcgtag agtttatttt tggtatctgg atattcagcc tccaagttcg tatgacgatt
     3841 aaatggctct tgaacaccgt gagtttcaag atctgcctca tcaatgaccc agaagcctag
     3901 tttatcgaag aggtcataca ccttaggatg gtttggataa tgcgagttac gaacagcatt
     3961 gatgttaaac ttcttcatta gaatcaagtc tctaacaaca aaatctaatg gcacagctct
     4021 accgaacctt ggatggtgat catgcctgtt gacacctcta aagagaatgt ctttgccatt
     4081 aacagtaatg ttaccgtcct tcaactccac ttgtctgaaa ccaacatggt gcttaataga
     4141 ttgaatcaca ctgccatcag atccaattaa atccaactgg tacttgtaca aagtaggatt
     4201 ttctgcggtc caatgttctg gggccttgac gttgatcttg aaagctgttt cttcgtcctt
     4261 tttggtcgaa aaggaaataa attctttagt tgaaaacgtc gtgttcccat tctcctcgtt
     4321 caacaaagag cttgcatcgt aaactttaga tccatcttca ggttcgtaaa gtgtgaaatt
     4381 gatgtgatca taagaagaac cctggacatc aactttcaca gaaagctctg catcctgata
     4441 ctgagagtcc acaaaagttg tagtgaccct aacgtcttca atatgggcct tcggcgcgcc
     4501 tcagccgagc ggccgcgtgt tacaaccaat taaccaattc tgattagaaa aactcatcga
     4561 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     4621 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     4681 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     4741 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     4801 gcaaaagctt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     4861 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgaggcgaa
     4921 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     4981 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     5041 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     5101 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     5161 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     5221 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     5281 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctggag caagacgttt
     5341 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     5401 ttgttcatga tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacaa
     5461 cgtggctttg ttgaataaat cgaacttttg ctgagttgaa ggatcagtca tgaccaaaat
     5521 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     5581 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     5641 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     5701 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     5761 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     5821 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     5881 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     5941 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6001 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6061 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6121 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6181 caacgcggcc tttttacggt tcctggcctt ttg
//
