LOCUS       sequence-259430-        6131 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LEU2 preassembled entry vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6131)
  AUTHORS   An-Adirekkun JM, Stewart CJ, Geller SH, Patel MT, Melendez J, Oakes 
            BL, Noyes MB, McClean MN
  TITLE     A yeast optogenetic toolkit (yOTK) for gene expression control in 
            Saccharomyces cerevisiae.
  JOURNAL   Biotechnol Bioeng. 2019 Dec 2. doi: 10.1002/bit.27234.
  PUBMED    31788779
REFERENCE   2  (bases 1 to 6131)
  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: "Biotechnol 
            Bioeng. 2019 Dec 2. doi: 10.1002/bit.27234."
COMMENT     Sequence Label: pMM0494
FEATURES             Location/Qualifiers
     source          1..6131
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             817..828
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             835..1548
                     /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      1560..1631
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1647..1674
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        2105..2512
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2513..3607
                     /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(4335..5150)
                     /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(4335..5150)
                     /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     5058..5077
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5142..5161
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5375..5963
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5864..5883
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctaaccgatg ccgaagaact gcgcagtccg gttataacgt ctgacatgtc cttttttgat
       61 ttggaatcca accactcaag tgactctgtt catttacttt gcgaaaaata tacccacaaa
      121 ttgcccatcg aaagtgaatc gcaaaccacc ttcagactgg caccgacaaa gcaaagatta
      181 tacagacaga gtactttata cgtaccgtta agtctcaagc aaagggtttt cttatttact
      241 gaacgggtaa agagtatctg ggccggcttg ccaagatgca aaccgaataa gtatttcaaa
      301 gttgcatttg ccttagccgt cctgacacca ttggctattt ggatatttta tattgacttt
      361 cgtgtacatt gatcacatcg actgttctat tggcaaatga accacgggca ttgactattt
      421 ttcaggttac tactatatat tatcatcacg ggcaaggatt gtaccctgaa ttcgcatcta
      481 gatggtagag ccacaaacag ccggtacaag caacgatctc caggaccatc tgaatcatgc
      541 gcggatgaca cgaactcacg acggcgatca cagacattaa cccacagtac agacactgcg
      601 acaacgtggc aattcgtcgc aataccgtct cactgaactg gccgataatt gcagacgaac
      661 gtgagaccga aagtgaaacg tgatttcatg cgtcattttg aacattttgt aaatcttatt
      721 taataatgtg tgcggcaatt cacatttaat ttatgaatgt tttcttaaca tcgcggcaac
      781 tcaagaaacg gcaggttcgg atcttagcta ctagagaaag aggagaaata ctagatgcgt
      841 aaaggcgaag agctgttcac tggtgtcgtc cctattctgg tggaactgga tggtgatgtc
      901 aacggtcata agttttccgt gcgtggcgag ggtgaaggtg acgcaactaa tggtaaactg
      961 acgctgaagt tcatctgtac tactggtaaa ctgccggttc cttggccgac tctggtaacg
     1021 acgctgactt atggtgttca gtgctttgct cgttatccgg accatatgaa gcagcatgac
     1081 ttcttcaagt ccgccatgcc ggaaggctat gtgcaggaac gcacgatttc ctttaaggat
     1141 gacggcacgt acaaaacgcg tgcggaagtg aaatttgaag gcgataccct ggtaaaccgc
     1201 attgagctga aaggcattga ctttaaagag gacggcaata tcctgggcca taagctggaa
     1261 tacaatttta acagccacaa tgtttacatc accgccgata aacaaaaaaa tggcattaaa
     1321 gcgaatttta aaattcgcca caacgtggag gatggcagcg tgcagctggc tgatcactac
     1381 cagcaaaaca ctccaatcgg tgatggtcct gttctgctgc cagacaatca ctatctgagc
     1441 acgcaaagcg ttctgtctaa agatccgaac gagaaacgcg atcatatggt tctgctggag
     1501 ttcgtaaccg cagcgggcat cacgcatggt atggatgaac tgtacaaatg accaggcatc
     1561 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     1621 tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc tgcgtttata
     1681 ggtctcagct gccaatgaga cgacggggtc atcacggctc atcatgcgcc aaacaaatgt
     1741 gtgcaataca cgctcggatg actgcatgat gaccgcactg actggggaca gcagatccac
     1801 ctaagcctgt gagagaagca gacacccgac agatcaaggc agttaactag tgcactgcag
     1861 tacatcgagg agaacttcta gtatatctac atacctaata ttattgcctt attaaaaatg
     1921 gaatcccaac aattacatca aaatccacat tctcttcaaa atcaattgtc ctgtacttcc
     1981 ttgttcatgt gtgttcaaaa acgttatatt tataggataa ttatactcta tttctcaaca
     2041 agtaattggt tgtttggccg agcggtctaa ggcgcctgat tcaagaaata tcttgaccgc
     2101 agttaactgt gggaatactc aggtatcgta agatgcaaga gttcgaatct cttagcaacc
     2161 attatttttt tcctcaacat aacgagaaca cacaggggcg ctatcgcaca gaatcaaatt
     2221 cgatgactgg aaattttttg ttaatttcag aggtcgcctg acgcatatac ctttttcaac
     2281 tgaaaaattg ggagaaaaag gaaaggtgag agcgccggaa ccggcttttc atatagaata
     2341 gagaagcgtt catgactaaa tgcttgcatc acaatacttg aagttgacaa tattatttaa
     2401 ggacctattg ttttttccaa taggtggtta gcaatcgtct tactttctaa cttttcttac
     2461 cttttacatt tcagcaatat atatatatat atttcaagga tataccattc taatgtctgc
     2521 ccctaagaag atcgtcgttt tgccaggtga ccacgttggt caagaaatca cagccgaagc
     2581 cattaaggtt cttaaagcta tttctgatgt tcgttccaat gtcaagttcg atttcgaaaa
     2641 tcatttaatt ggtggtgctg ctatcgatgc tacaggtgtt ccacttccag atgaggcgct
     2701 ggaagcctcc aagaaggctg atgccgtttt gttaggtgct gtgggtggtc ctaaatgggg
     2761 aaccggtagt gttagacctg aacaaggttt actaaaaatc cgtaaagaac ttcaattgta
     2821 cgccaactta agaccatgta actttgcatc cgactctctt ttagacttat ctccaatcaa
     2881 gccacaattt gctaaaggta ctgacttcgt tgttgtcaga gaattagtgg gaggtattta
     2941 ctttggtaag agaaaggagg acgatggtga tggtgtcgct tgggatagtg aacaatacac
     3001 cgttccagaa gtgcaaagaa tcacaagaat ggccgctttc atggccctac aacatgagcc
     3061 accattgcct atttggtcct tggataaagc taatgttttg gcctcttcaa gattatggag
     3121 aaaaactgtg gaggaaacca tcaagaacga atttcctaca ttgaaggttc aacatcaatt
     3181 gattgattct gccgccatga tcctagttaa gaacccaacc cacctaaatg gtattataat
     3241 caccagcaac atgtttggtg atatcatctc cgatgaagcc tccgttatcc caggttcctt
     3301 gggtttgttg ccatctgcgt ccttggcctc tttgccagac aagaacaccg catttggttt
     3361 gtacgaacca tgccacggtt ctgctccaga tttgccaaag aataaggtca accctatcgc
     3421 cactatcttg tctgctgcaa tgatgttgaa attgtcattg aacttgcctg aagaaggtaa
     3481 ggccattgaa gatgcagtta aaaaggtttt ggatgcaggt atcagaactg gtgatttagg
     3541 tggttccaac agtaccaccg aagtcggtga tgctgtcgcc gaagaagtta agaaaatcct
     3601 tgcttaacct ggaggaccct tctctttaga ctattctact cttatgcacg taaaaaattc
     3661 taggaaatat gtattaacta ggagtaaaat aaccggctag tggcattcat atagccgtct
     3721 gtttacatct acatcacaca tttcgagtgt atatctcgca acgttggcgt taaataggca
     3781 ggagtactcg tatcgcatgt cggtgcgaca cgaaattaca aaatggaata tgttcatagg
     3841 gtagacgaaa ctatatacgc aatctacata catttatcaa gaaggagaaa aaggaggatg
     3901 taaaggaata caggtaagca aattgatact aatggctcaa cgtgataagg aaaaagaatt
     3961 gcactttaac attaatattg acaaggagga gggcaccaca caaaaagtta ggtgtaacag
     4021 aaaatcatga aactatgatt cctaatttat atattggagg attttctcta aaaaaaaaaa
     4081 aatacaacaa ataaaaaaca ctcaatgacc tgaccatttg atggagttta agtcaatacc
     4141 ttcttgaacc atttcccata atggtgaaag ttccctcaag aattttactc tgtcagaaac
     4201 ggccttaacg acgtagtcga cctcctcttc agtactaaat ctaccaatac caaatctgat
     4261 ggaagaatgg gctaatgcat catccttacc cagcgccgag cggccgcgtg ttacaaccaa
     4321 ttaaccaatt ctgattagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata
     4381 tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca
     4441 ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca
     4501 acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca
     4561 ccatgagtga cgactgaatc cggtgagaat ggcaaaagct tatgcatttc tttccagact
     4621 tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta
     4681 ttcattcgtg attgcgcctg agcgaggcga aatacgcgat cgctgttaaa aggacaatta
     4741 caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca
     4801 cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg
     4861 agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat
     4921 tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg
     4981 ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca
     5041 cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg
     5101 gaatttaatc gcggcctgga gcaagacgtt tcccgttgaa tatggctcat aacacccctt
     5161 gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt tttatcttgt
     5221 gcaatgtaac atcagagatt ttgagacaca acgtggcttt gttgaataaa tcgaactttt
     5281 gctgagttga aggatcagtc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5341 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5401 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5461 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5521 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5581 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5641 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5701 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     5761 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     5821 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     5881 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5941 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6001 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     6061 gtgcggccgc caatacccag aagaacagta aaataaagca aggtacgtga aattaatatt
     6121 tttaaatggt t
//
