LOCUS       sequence-318051-        5928 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting the LAC4 locus. 
            Histidine selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5928)
  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 5928)
  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-HIS
FEATURES             Location/Qualifiers
     source          1..5928
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      706..777
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      793..820
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        1081..1268
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             1269..1931
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     CDS             complement(3076..3891)
                     /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(3076..3891)
                     /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     3799..3818
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3883..3902
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4116..4704
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4605..4624
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     RBS             5891..5902
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(5909..5928,1..694)
                     /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"
ORIGIN
        1 gttcactggt gtcgtcccta ttctggtgga actggatggt gatgtcaacg gtcataagtt
       61 ttccgtgcgt ggcgagggtg aaggtgacgc aactaatggt aaactgacgc tgaagttcat
      121 ctgtactact ggtaaactgc cggttccttg gccgactctg gtaacgacgc tgacttatgg
      181 tgttcagtgc tttgctcgtt atccggacca tatgaagcag catgacttct tcaagtccgc
      241 catgccggaa ggctatgtgc aggaacgcac gatttccttt aaggatgacg gcacgtacaa
      301 aacgcgtgcg gaagtgaaat ttgaaggcga taccctggta aaccgcattg agctgaaagg
      361 cattgacttt aaagaggacg gcaatatcct gggccataag ctggaataca attttaacag
      421 ccacaatgtt tacatcaccg ccgataaaca aaaaaatggc attaaagcga attttaaaat
      481 tcgccacaac gtggaggatg gcagcgtgca gctggctgat cactaccagc aaaacactcc
      541 aatcggtgat ggtcctgttc tgctgccaga caatcactat ctgagcacgc aaagcgttct
      601 gtctaaagat ccgaacgaga aacgcgatca tatggttctg ctggagttcg taaccgcagc
      661 gggcatcacg catggtatgg atgaactgta caaatgacca ggcatcaaat aaaacgaaag
      721 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac
      781 tagagtcaca ctggctcacc ttcgggtggg cctttctgcg tttataggtc tcagctggaa
      841 atctgctcgt cagtggtgct cacactgacg aatcatgtac agatcatacc gatgactgcc
      901 tggcgactca caactaagca agacagccgg aaccagcgcc ggcgaacacc actgcatata
      961 tggcatatca caacagtcca cgtctcaagc agttacagag atgttacgaa ccactagtgc
     1021 actgcagtac aaacacagtc ctttcccgca attttctttt tctattactc ttggcctcct
     1081 ctagtacact ctatattttt ttatgcctcg gtaatgattt tcattttttt ttttccacct
     1141 agcggatgac tctttttttt tcttagcgat tggcattatc acataatgaa ttatacatta
     1201 tataaagtaa tgtgatttct tcgaagaata tactaaaaaa tgagcaggca agataaacga
     1261 aggcaaagat gacagagcag aaagccctag taaagcgtat tacaaatgaa accaagattc
     1321 agattgcgat ctctttaaag ggtggtcccc tagcgataga gcactcgatc ttcccagaaa
     1381 aagaggcaga agcagtagca gaacaggcca cacaatcgca agtgattaac gtccacacag
     1441 gtatagggtt tctggaccat atgatacatg ctctggccaa gcattccggc tggtcgctaa
     1501 tcgttgagtg cattggtgac ttacacatag acgaccatca caccactgag gactgcggga
     1561 ttgctctcgg tcaagctttt aaagaggccc taggggccgt gcgtggagta aaaaggtttg
     1621 gatcaggatt tgcgcctttg gatgaggcac tttccagagc ggtggttgat ctttcgaaca
     1681 ggccgtacgc agttgtcgaa cttggtttgc aaagggagaa agtaggtgat ctctcttgcg
     1741 agatgatccc gcattttctt gaaagctttg cagaggctag cagaattacc ctccacgttg
     1801 attgtctgcg aggcaagaat gatcatcacc gtagtgagag tgcgttcaag gctcttgcgg
     1861 ttgccataag agaagccacc tcgcccaatg gtaccaacga tgttccctcc accaaaggtg
     1921 ttcttatgta gtgacaccga ttatttaaag ctgctgcata cgatatatat acatgtgtat
     1981 atatgtatac ctatgaatgt cagtaagtat gtatacgaac agtatgatac tgaagatgac
     2041 aaggtaatgc atcattctat acgtgtcatt ctgaacgagg cgcgctttcc ttttttcttt
     2101 ttgctttttc tttttttttc tcttgaactc gacggatcat agagttgctt aattagcttg
     2161 tacatggctt tgtggtttct gccataacaa gcttcgttac ccaaggacca gataataatc
     2221 gaaggatgat tgacatctct taggacaagt tgggaagctc tgtctaagta cgcgacctcg
     2281 tactctggat tatctgataa gtaatgggca ttaacatcgt agagtttatt tttggtatct
     2341 ggatattcag cctccaagtt cgtatgacga ttaaatggct cttgaacacc gtgagtttca
     2401 agatctgcct catcaatgac ccagaagcct agtttatcga agaggtcata caccttagga
     2461 tggtttggat aatgcgagtt acgaacagca ttgatgttaa acttcttcat tagaatcaag
     2521 tctctaacaa caaaatctaa tggcacagct ctaccgaacc ttggatggtg atcatgcctg
     2581 ttgacacctc taaagagaat gtctttgcca ttaacagtaa tgttaccgtc cttcaactcc
     2641 acttgtctga aaccaacatg gtgcttaata gattgaatca cactgccatc agatccaatt
     2701 aaatccaact ggtacttgta caaagtagga ttttctgcgg tccaatgttc tggggccttg
     2761 acgttgatct tgaaagctgt ttcttcgtcc tttttggtcg aaaaggaaat aaattcttta
     2821 gttgaaaacg tcgtgttccc attctcctcg ttcaacaaag agcttgcatc gtaaacttta
     2881 gatccatctt caggttcgta aagtgtgaaa ttgatgtgat cataagaaga accctggaca
     2941 tcaactttca cagaaagctc tgcatcctga tactgagagt ccacaaaagt tgtagtgacc
     3001 ctaacgtctt caatatgggc cttcggcgcg cctcagccga gcggccgcgt gttacaacca
     3061 attaaccaat tctgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     3121 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     3181 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     3241 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     3301 accatgagtg acgactgaat ccggtgagaa tggcaaaagc ttatgcattt ctttccagac
     3361 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     3421 attcattcgt gattgcgcct gagcgaggcg aaatacgcga tcgctgttaa aaggacaatt
     3481 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     3541 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     3601 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     3661 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     3721 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     3781 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     3841 ggaatttaat cgcggcctgg agcaagacgt ttcccgttga atatggctca taacacccct
     3901 tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat ttttatcttg
     3961 tgcaatgtaa catcagagat tttgagacac aacgtggctt tgttgaataa atcgaacttt
     4021 tgctgagttg aaggatcagt catgaccaaa atcccttaac gtgagttttc gttccactga
     4081 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4141 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4201 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     4261 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     4321 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     4381 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     4441 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     4501 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     4561 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4621 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     4681 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     4741 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     4801 cgtgcggccg cccctttcag gcgcgccgaa agtgattgaa gaaccctcaa acttgctgga
     4861 tatttcctta cccttgacct ttaggctttc aattttaccc aacaatttgt ccaagacaaa
     4921 ttgcaatcca ctggattcaa ctgagacata acgtttaccg tcgttgatct tggtaggttt
     4981 ttctgctgtc tctgtaacaa aatctggtac ctttaatgga agttcggctt ggccccaggc
     5041 aatttcatga cctgccttta gaacaccagc atcatccttt aacacggcaa caacataagt
     5101 ggtatcagaa ggaatagtaa cagattcttc tggctttaaa gctggaacgt cgattgtctt
     5161 tcccgtgtcc ttgtcgataa acaataagtg atctgtcgta atgaagtcgt gcttatttgt
     5221 gattgttaca gatccgtgcg caattttaat atgaacgggt tcaataacct tcttatactc
     5281 tacaaggccc ggagtaggat tatgctcact gttacacaaa ccatccatga tgaacactcc
     5341 gtcatgaacc tcttccttaa agtcaccacc ataagcataa gctttatgca acttaccatc
     5401 tgcagtacta acatcttcga attcaatacc gtgatttgcc cattcccaga taaagccacc
     5461 ttggtaaaac ttctccttgt agaacaactc ttgatattct ttcaaagaac ccgggccgtt
     5521 acccattgca tgaccgtact cacacaagat caaaggcttt tcaaacttac cattttcatc
     5581 agtgtggttc ttcctccacc tttccataat ttcaaatgtt gggtacatga aactaaagat
     5641 atctgcactc aaagcgttca agtcaccctc ataatgcacc agtctggtag gatccaattg
     5701 cttaagaatt cgcatctaga ctgatgagac gaacgtgaga ccgaaagtga aacgtgattt
     5761 catgcgtcat tttgaacatt ttgtaaatct tatttaataa tgtgtgcggc aattcacatt
     5821 taatttatga atgttttctt aacatcgcgg caactcaaga aacggcaggt tcggatctta
     5881 gctactagag aaagaggaga aatactagat gcgtaaaggc gaagagct
//
