LOCUS       sequence-318047-        5819 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrative vector for K. marxianus, targeting the ARO3 locus. 
            Uracil selection. Low integration efficiency in an NHEJ-deficient 
            background..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5819)
  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 5819)
  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: pI6-MTU-DO-URA
FEATURES             Location/Qualifiers
     source          1..5819
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(113..124)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     RBS             1068..1079
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1086..1799
                     /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      1811..1882
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1898..1925
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             2314..3117
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     CDS             complement(4099..4914)
                     /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(4099..4914)
                     /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     4822..4841
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4906..4925
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5139..5727
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5628..5647
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taaccgtgcg gccgcccctt tcaggcgcgc ctaaggcagt agagcagtag caaagagcaa
       61 aaccaaagga aaaaaaaaat aaaaataaaa aaagggaagg gaaaccgacc caccgacctt
      121 caattcgctg acataatgct catatattct acaaccactc tacgtaataa attccacgat
      181 gtactttata ctatttcccc tgcgacaagg gcgccaacgc tgctattacc accactatac
      241 agaatataat ataatatagt atagtacaat atatggtgca tatgaccccc atgtggctta
      301 tataatcaaa atgaaaatgg tgtcacgtga ctctaattac ttcttggcgt gttttcggag
      361 tagataaaca ggggcgataa acaggggcat atacgggggc cagcgagcga gtagtgccac
      421 ggccccttta aacactgggg tatgccttca agaaccaaaa aaaaaattgc caattcctaa
      481 tatcgtaata ttcaagaata caagcatatt caaattttgc ggagttataa ctcagctggc
      541 accaattaca tccacgtgac ccgatccagc gccggattaa ctttccctag aaaaaatttt
      601 ttttttttat tttttttcca atttctccat tgtttctgcc aaattactca taatgccaca
      661 ttttagaaat ttttcaggtt cggtgagatc aagccggtta cgcacaaaaa gcttgaaaaa
      721 ttcaggaacc gtggtatata atgaatcatc gcatgatgag agagagagaa gaaagagaag
      781 aataaataat aaccattaag agttgttgtt gtttcattat ttcagttgca ttccttttgt
      841 atttttttct tgttagatag gtaactaacg gtaataagtg tagaattcgc atctagactg
      901 atgagacgaa cgtgagaccg aaagtgaaac gtgatttcat gcgtcatttt gaacattttg
      961 taaatcttat ttaataatgt gtgcggcaat tcacatttaa tttatgaatg ttttcttaac
     1021 atcgcggcaa ctcaagaaac ggcaggttcg gatcttagct actagagaaa gaggagaaat
     1081 actagatgcg taaaggcgaa gagctgttca ctggtgtcgt ccctattctg gtggaactgg
     1141 atggtgatgt caacggtcat aagttttccg tgcgtggcga gggtgaaggt gacgcaacta
     1201 atggtaaact gacgctgaag ttcatctgta ctactggtaa actgccggtt ccttggccga
     1261 ctctggtaac gacgctgact tatggtgttc agtgctttgc tcgttatccg gaccatatga
     1321 agcagcatga cttcttcaag tccgccatgc cggaaggcta tgtgcaggaa cgcacgattt
     1381 cctttaagga tgacggcacg tacaaaacgc gtgcggaagt gaaatttgaa ggcgataccc
     1441 tggtaaaccg cattgagctg aaaggcattg actttaaaga ggacggcaat atcctgggcc
     1501 ataagctgga atacaatttt aacagccaca atgtttacat caccgccgat aaacaaaaaa
     1561 atggcattaa agcgaatttt aaaattcgcc acaacgtgga ggatggcagc gtgcagctgg
     1621 ctgatcacta ccagcaaaac actccaatcg gtgatggtcc tgttctgctg ccagacaatc
     1681 actatctgag cacgcaaagc gttctgtcta aagatccgaa cgagaaacgc gatcatatgg
     1741 ttctgctgga gttcgtaacc gcagcgggca tcacgcatgg tatggatgaa ctgtacaaat
     1801 gaccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     1861 ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt
     1921 ctgcgtttat aggtctcagc tggaaatctg ctcgtcagtg gtgctcacac tgacgaatca
     1981 tgtacagatc ataccgatga ctgcctggcg actcacaact aagcaagaca gccggaacca
     2041 gcgccggcga acaccactgc atatatggca tatcacaaca gtccacgtct caagcagtta
     2101 cagagatgtt acgaaccact agtgcactgc agtacacggt ttccttgaaa tttttttgat
     2161 tcggtaatct ccgaacagaa ggaagaacga aggaaggagc acagacttag attggtatat
     2221 atacgcatat gtagtgttga agaaacatga aattgcccag tattcttaac ccaactgcac
     2281 agaacaaaaa cgtgcaggaa acgaagataa atcatgtcga aagctacata taaggaacgt
     2341 gctgctactc atcctagtcc tgttgctgcc aagctattta atatcatgca cgaaaagcaa
     2401 acaaacttgt gtgcttcatt ggatgttcgt accaccaagg aattactgga gttagttgaa
     2461 gcattaggtc ccaaaatttg tttactaaaa acacatgtgg atatcttgac tgatttttcc
     2521 atggagggca cagttaagcc gctaaaggca ttatccgcca agtacaattt tttactcttc
     2581 gaggacagaa aatttgctga cattggtaat acagtcaaat tgcagtactc tgcgggtgta
     2641 tacagaatag cagaatgggc agacattacg aatgcacacg gtgtggtggg cccaggtatt
     2701 gttagcggtt tgaagcaggc ggcagaagaa gtaacaaagg aacctagagg ccttttgatg
     2761 ttagcagaat tgtcatgcaa gggctcccta tctactggag aatatactaa gggtactgtt
     2821 gacattgcga agagcgacaa agattttgtt atcggcttta ttgctcaaag agacatgggt
     2881 ggaagagatg aaggttacga ttggttgatt atgacacccg gtgtgggttt agatgacaag
     2941 ggagatgcat tgggtcaaca gtatagaacc gtggatgatg tggtttctac aggatctgac
     3001 attattattg ttggaagagg actatttgca aagggaaggg atgctaaggt agagggtgaa
     3061 cgttacagaa aagcaggctg ggaagcatat ttgagaagat gcggccagca aaactaaaaa
     3121 actgtattat aagtaaatgc atgtatacta aactcacaaa ttagagcttc aatttaatta
     3181 tatcagttat tacccgagta ggctaaggtt gttgatgctg aaggtgttca aagaagaatt
     3241 atgattgatt gctctcatgg taactccagc aaagacttca gaaatcagcc aaaggttgcc
     3301 caatccatct acgaacaact taccgatgga caaaatgcta tctgcggtgt aatgatcgag
     3361 tctaacttgg tagaaggcag ataagatatt cctccagaag gtggccgtga caaattgatc
     3421 tacggatgtt caattactga tgcttgtatt ggttgggata ccactgatga ggtacttcgt
     3481 ctattggcca aaggtgtcca aaatagaaga gcagcattga agaaataagt caactaaaac
     3541 tcttttataa aacaaaacaa acaaaatgac aaattaatcc cgattaattt aggcgtcctt
     3601 cctgtagttg cttgccaaaa gacgggcgag agcccagaat aaatcaaaaa ctatttatcc
     3661 tcttggtatg gaacgtatat atgtatacat atataataat aattatctat tttataaaga
     3721 tgaaaagttc aatttcatac attctattct agtgtgaaag tgtacatatg tataaaagcg
     3781 aaaattgatg ttgagctgat cgaatgtttg tatgatagca agtttttctt ctttctcctt
     3841 ttgtttgcta gaacgatgtg gcattataag attattgttt gcaaagcgga atttaaccta
     3901 ttgaaagtga ttcttcactc ataaaaggta aaaaaaaaaa agaagagaga gagatattga
     3961 aaatgtggaa tgaagactat tagtatagta gaattagaat taaaattgaa actcagttca
     4021 tgtagaatca gttgtaatag tgttgttggc gcgcctcagc cgagcggccg cgtgttacaa
     4081 ccaattaacc aattctgatt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     4141 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     4201 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     4261 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     4321 atcaccatga gtgacgactg aatccggtga gaatggcaaa agcttatgca tttctttcca
     4381 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     4441 gttattcatt cgtgattgcg cctgagcgag gcgaaatacg cgatcgctgt taaaaggaca
     4501 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     4561 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     4621 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     4681 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     4741 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     4801 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     4861 gttggaattt aatcgcggcc tggagcaaga cgtttcccgt tgaatatggc tcataacacc
     4921 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgatgata tatttttatc
     4981 ttgtgcaatg taacatcaga gattttgaga cacaacgtgg ctttgttgaa taaatcgaac
     5041 ttttgctgag ttgaaggatc agtcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5101 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5161 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5221 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5281 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5341 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5401 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5461 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5521 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5581 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5641 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5701 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5761 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtgga
//
