LOCUS       sequence-317739-        6735 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting the LAC4 locus. 
            Leucine selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6735)
  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 6735)
  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-LEU
FEATURES             Location/Qualifiers
     source          1..6735
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             1095..1106
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1113..1826
                     /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      1838..1909
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1925..1952
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        2404..2811
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2812..3906
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     CDS             complement(5015..5830)
                     /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(5015..5830)
                     /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     5738..5757
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5822..5841
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      6055..6643
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6544..6563
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taaccgtgcg gccgcccctt tcaggcgcgc cgaaagtgat tgaagaaccc tcaaacttgc
       61 tggatatttc cttacccttg acctttaggc tttcaatttt acccaacaat ttgtccaaga
      121 caaattgcaa tccactggat tcaactgaga cataacgttt accgtcgttg atcttggtag
      181 gtttttctgc tgtctctgta acaaaatctg gtacctttaa tggaagttcg gcttggcccc
      241 aggcaatttc atgacctgcc tttagaacac cagcatcatc ctttaacacg gcaacaacat
      301 aagtggtatc agaaggaata gtaacagatt cttctggctt taaagctgga acgtcgattg
      361 tctttcccgt gtccttgtcg ataaacaata agtgatctgt cgtaatgaag tcgtgcttat
      421 ttgtgattgt tacagatccg tgcgcaattt taatatgaac gggttcaata accttcttat
      481 actctacaag gcccggagta ggattatgct cactgttaca caaaccatcc atgatgaaca
      541 ctccgtcatg aacctcttcc ttaaagtcac caccataagc ataagcttta tgcaacttac
      601 catctgcagt actaacatct tcgaattcaa taccgtgatt tgcccattcc cagataaagc
      661 caccttggta aaacttctcc ttgtagaaca actcttgata ttctttcaaa gaacccgggc
      721 cgttacccat tgcatgaccg tactcacaca agatcaaagg cttttcaaac ttaccatttt
      781 catcagtgtg gttcttcctc cacctttcca taatttcaaa tgttgggtac atgaaactaa
      841 agatatctgc actcaaagcg ttcaagtcac cctcataatg caccagtctg gtaggatcca
      901 attgcttaag aattcgcatc tagactgatg agacgaacgt gagaccgaaa gtgaaacgtg
      961 atttcatgcg tcattttgaa cattttgtaa atcttattta ataatgtgtg cggcaattca
     1021 catttaattt atgaatgttt tcttaacatc gcggcaactc aagaaacggc aggttcggat
     1081 cttagctact agagaaagag gagaaatact agatgcgtaa aggcgaagag ctgttcactg
     1141 gtgtcgtccc tattctggtg gaactggatg gtgatgtcaa cggtcataag ttttccgtgc
     1201 gtggcgaggg tgaaggtgac gcaactaatg gtaaactgac gctgaagttc atctgtacta
     1261 ctggtaaact gccggttcct tggccgactc tggtaacgac gctgacttat ggtgttcagt
     1321 gctttgctcg ttatccggac catatgaagc agcatgactt cttcaagtcc gccatgccgg
     1381 aaggctatgt gcaggaacgc acgatttcct ttaaggatga cggcacgtac aaaacgcgtg
     1441 cggaagtgaa atttgaaggc gataccctgg taaaccgcat tgagctgaaa ggcattgact
     1501 ttaaagagga cggcaatatc ctgggccata agctggaata caattttaac agccacaatg
     1561 tttacatcac cgccgataaa caaaaaaatg gcattaaagc gaattttaaa attcgccaca
     1621 acgtggagga tggcagcgtg cagctggctg atcactacca gcaaaacact ccaatcggtg
     1681 atggtcctgt tctgctgcca gacaatcact atctgagcac gcaaagcgtt ctgtctaaag
     1741 atccgaacga gaaacgcgat catatggttc tgctggagtt cgtaaccgca gcgggcatca
     1801 cgcatggtat ggatgaactg tacaaatgac caggcatcaa ataaaacgaa aggctcagtc
     1861 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctct actagagtca
     1921 cactggctca ccttcgggtg ggcctttctg cgtttatagg tctcagctgg aaatctgctc
     1981 gtcagtggtg ctcacactga cgaatcatgt acagatcata ccgatgactg cctggcgact
     2041 cacaactaag caagacagcc ggaaccagcg ccggcgaaca ccactgcata tatggcatat
     2101 cacaacagtc cacgtctcaa gcagttacag agatgttacg aaccactagt gcactgcagt
     2161 acatcgagga gaacttctag tatatctaca tacctaatat tattgcctta ttaaaaatgg
     2221 aatcccaaca attacatcaa aatccacatt ctcttcaaaa tcaattgtcc tgtacttcct
     2281 tgttcatgtg tgttcaaaaa cgttatattt ataggataat tatactctat ttctcaacaa
     2341 gtaattggtt gtttggccga gcggtctaag gcgcctgatt caagaaatat cttgaccgca
     2401 gttaactgtg ggaatactca ggtatcgtaa gatgcaagag ttcgaatctc ttagcaacca
     2461 ttattttttt cctcaacata acgagaacac acaggggcgc tatcgcacag aatcaaattc
     2521 gatgactgga aattttttgt taatttcaga ggtcgcctga cgcatatacc tttttcaact
     2581 gaaaaattgg gagaaaaagg aaaggtgaga gcgccggaac cggcttttca tatagaatag
     2641 agaagcgttc atgactaaat gcttgcatca caatacttga agttgacaat attatttaag
     2701 gacctattgt tttttccaat aggtggttag caatcgtctt actttctaac ttttcttacc
     2761 ttttacattt cagcaatata tatatatata tttcaaggat ataccattct aatgtctgcc
     2821 cctaagaaga tcgtcgtttt gccaggtgac cacgttggtc aagaaatcac agccgaagcc
     2881 attaaggttc ttaaagctat ttctgatgtt cgttccaatg tcaagttcga tttcgaaaat
     2941 catttaattg gtggtgctgc tatcgatgct acaggtgttc cacttccaga tgaggcgctg
     3001 gaagcctcca agaaggctga tgccgttttg ttaggtgctg tgggtggtcc taaatgggga
     3061 accggtagtg ttagacctga acaaggttta ctaaaaatcc gtaaagaact tcaattgtac
     3121 gccaacttaa gaccatgtaa ctttgcatcc gactctcttt tagacttatc tccaatcaag
     3181 ccacaatttg ctaaaggtac tgacttcgtt gttgtcagag aattagtggg aggtatttac
     3241 tttggtaaga gaaaggagga cgatggtgat ggtgtcgctt gggatagtga acaatacacc
     3301 gttccagaag tgcaaagaat cacaagaatg gccgctttca tggccctaca acatgagcca
     3361 ccattgccta tttggtcctt ggataaagct aatgttttgg cctcttcaag attatggaga
     3421 aaaactgtgg aggaaaccat caagaacgaa tttcctacat tgaaggttca acatcaattg
     3481 attgattctg ccgccatgat cctagttaag aacccaaccc acctaaatgg tattataatc
     3541 accagcaaca tgtttggtga tatcatctcc gatgaagcct ccgttatccc aggttccttg
     3601 ggtttgttgc catctgcgtc cttggcctct ttgccagaca agaacaccgc atttggtttg
     3661 tacgaaccat gccacggttc tgctccagat ttgccaaaga ataaggtcaa ccctatcgcc
     3721 actatcttgt ctgctgcaat gatgttgaaa ttgtcattga acttgcctga agaaggtaag
     3781 gccattgaag atgcagttaa aaaggttttg gatgcaggta tcagaactgg tgatttaggt
     3841 ggttccaaca gtaccaccga agtcggtgat gctgtcgccg aagaagttaa gaaaatcctt
     3901 gcttaacctg gaggaccctt ctctttagac tattctactc ttatgcacgt aaaaaattct
     3961 aggaaatatg tattaactag gagtaaaata accggctagt ggcattcata tagccgtctg
     4021 tttacatcta catcacacat ttcgagtgta tatctcgcaa cgttggcgtt aaataggcag
     4081 gagttgctta attagcttgt acatggcttt gtggtttctg ccataacaag cttcgttacc
     4141 caaggaccag ataataatcg aaggatgatt gacatctctt aggacaagtt gggaagctct
     4201 gtctaagtac gcgacctcgt actctggatt atctgataag taatgggcat taacatcgta
     4261 gagtttattt ttggtatctg gatattcagc ctccaagttc gtatgacgat taaatggctc
     4321 ttgaacaccg tgagtttcaa gatctgcctc atcaatgacc cagaagccta gtttatcgaa
     4381 gaggtcatac accttaggat ggtttggata atgcgagtta cgaacagcat tgatgttaaa
     4441 cttcttcatt agaatcaagt ctctaacaac aaaatctaat ggcacagctc taccgaacct
     4501 tggatggtga tcatgcctgt tgacacctct aaagagaatg tctttgccat taacagtaat
     4561 gttaccgtcc ttcaactcca cttgtctgaa accaacatgg tgcttaatag attgaatcac
     4621 actgccatca gatccaatta aatccaactg gtacttgtac aaagtaggat tttctgcggt
     4681 ccaatgttct ggggccttga cgttgatctt gaaagctgtt tcttcgtcct ttttggtcga
     4741 aaaggaaata aattctttag ttgaaaacgt cgtgttccca ttctcctcgt tcaacaaaga
     4801 gcttgcatcg taaactttag atccatcttc aggttcgtaa agtgtgaaat tgatgtgatc
     4861 ataagaagaa ccctggacat caactttcac agaaagctct gcatcctgat actgagagtc
     4921 cacaaaagtt gtagtgaccc taacgtcttc aatatgggcc ttcggcgcgc ctcagccgag
     4981 cggccgcgtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg agcatcaaat
     5041 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     5101 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     5161 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     5221 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagct
     5281 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     5341 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgaggcga aatacgcgat
     5401 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     5461 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     5521 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     5581 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     5641 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     5701 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     5761 ataaatcagc atccatgttg gaatttaatc gcggcctgga gcaagacgtt tcccgttgaa
     5821 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     5881 atgatatatt tttatcttgt gcaatgtaac atcagagatt ttgagacaca acgtggcttt
     5941 gttgaataaa tcgaactttt gctgagttga aggatcagtc atgaccaaaa tcccttaacg
     6001 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6061 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6121 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6181 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6241 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6301 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6361 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6421 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     6481 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     6541 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     6601 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     6661 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     6721 ccctgattct gtgga
//
