LOCUS       sequence-259428-        7396 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LEU2 integrtion vector for VP16-CIB1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7396)
  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 7396)
  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: pMM0554
FEATURES             Location/Qualifiers
     source          1..7396
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             37..57
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     285..304
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        285..303
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             328..837
                     /codon_start=1
                     /product="N-terminal portion of the CIB1 transcription 
                     factor from Arabidopsis thaliana"
                     /label=CIBN
                     /note="binds to CRY2 (cryptochrome 2) that has absorbed 
                     blue light (Kennedy et al., 2010)"
                     /translation="MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSS
                     MITGGEMDSYLSTAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCN
                     EKKKKMTMNRDDLVEEGEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKE
                     LEKTDYI"
     terminator      1387..1574
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(1442..1466)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        2029..2436
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2437..3531
                     /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(4259..5074)
                     /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(4259..5074)
                     /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     4982..5001
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5066..5085
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      5299..5887
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5788..5807
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        6730..7396
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     7266..7289
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
ORIGIN
        1 agatctatgg ataaagcgga attaattccc gagcctccaa aaaagaagag aaaggtcgaa
       61 ttgggtaccg ccgcctcgga gctccactta gacggcgagg acgtggcgat ggcgcatgcc
      121 gacgcgctag acgatttcga tctggacatg ttgggggacg gggattcccc gggtccggga
      181 tttacccccc acgactccgc cccctacggc gctctggata tggccgactt cgagtttgag
      241 cagatgttta ccgatgccct tggaattgac gagtacggtg ggtttaatac gactcactat
      301 agggcgagcg ccgaagctag cgccaccatg aatggagcta taggaggtga ccttttgctc
      361 aattttcctg acatgtcggt cctagagcgc caaagggctc acctcaagta cctcaatccc
      421 acctttgatt ctcctctcgc cggcttcttt gccgattctt caatgattac cggcggcgag
      481 atggacagct atctttcgac tgccggtttg aatcttccga tgatgtacgg tgaaacgacg
      541 gtggaaggtg attcaagact ctcaatttcg ccggaaacga cgcttgggac tggaaatttc
      601 aagaaacgga agtttgatac agagactaag gattgtaatg agaagaagaa gaagatgacg
      661 atgaacagag atgacctagt agaagaagga gaagaagaga agtcgaaaat aacagagcaa
      721 aacaatggga gcacaaaaag catcaagaag atgaaacaca aagccaagaa agaagagaac
      781 aatttctcta atgattcatc taaagtgacg aaggaattgg agaaaacgga ttatattcat
      841 gttcgtgcac gacgaggcca agccactgat agtcacagca tagcagaacg agttagaaga
      901 gaaaagatca gtgagagaat gaagtttcta caagatttgg ttcctggatg cgacaagatc
      961 acaggcaaag cagggatgct tgatgaaatc attaactatg ttcagtctct tcagagacaa
     1021 atcgagttct tatcgatgaa actagcaatt gtgaatccaa ggccggattt tgatatggat
     1081 gacatttttg ccaaagaggt tgcctcaact ccaatgactg tggtgccatc tcctgaaatg
     1141 gttctttccg gttattctca tgagatggtt cactctggtt attctagtga gatggttaac
     1201 tccggttacc ttcatgtcaa tccaatgcag caagtgaata ccagttctga tccattgtca
     1261 tgcttcaaca atggcgaagc tccttcgatg tgggactctc atgtgcagaa tctctatggc
     1321 aatttaggag taccggtcat cgagctcgag ctgcagatga atcgtagata cggatcctaa
     1381 ctcgaggcga atttcttatg atttatgatt tttattatta aataagttat aaaaaaaata
     1441 agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt
     1501 aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc tcttattgac
     1561 cacacctcta ccggcatgcc gagcaaatgc ctgcaaatcg ctccccattt cgctgccaat
     1621 gagacgacgg ggtcatcacg gctcatcatg cgccaaacaa atgtgtgcaa tacacgctcg
     1681 gatgactgca tgatgaccgc actgactggg gacagcagat ccacctaagc ctgtgagaga
     1741 agcagacacc cgacagatca aggcagttaa ctagtgcact gcagtacatc gaggagaact
     1801 tctagtatat ctacatacct aatattattg ccttattaaa aatggaatcc caacaattac
     1861 atcaaaatcc acattctctt caaaatcaat tgtcctgtac ttccttgttc atgtgtgttc
     1921 aaaaacgtta tatttatagg ataattatac tctatttctc aacaagtaat tggttgtttg
     1981 gccgagcggt ctaaggcgcc tgattcaaga aatatcttga ccgcagttaa ctgtgggaat
     2041 actcaggtat cgtaagatgc aagagttcga atctcttagc aaccattatt tttttcctca
     2101 acataacgag aacacacagg ggcgctatcg cacagaatca aattcgatga ctggaaattt
     2161 tttgttaatt tcagaggtcg cctgacgcat ataccttttt caactgaaaa attgggagaa
     2221 aaaggaaagg tgagagcgcc ggaaccggct tttcatatag aatagagaag cgttcatgac
     2281 taaatgcttg catcacaata cttgaagttg acaatattat ttaaggacct attgtttttt
     2341 ccaataggtg gttagcaatc gtcttacttt ctaacttttc ttacctttta catttcagca
     2401 atatatatat atatatttca aggatatacc attctaatgt ctgcccctaa gaagatcgtc
     2461 gttttgccag gtgaccacgt tggtcaagaa atcacagccg aagccattaa ggttcttaaa
     2521 gctatttctg atgttcgttc caatgtcaag ttcgatttcg aaaatcattt aattggtggt
     2581 gctgctatcg atgctacagg tgttccactt ccagatgagg cgctggaagc ctccaagaag
     2641 gctgatgccg ttttgttagg tgctgtgggt ggtcctaaat ggggaaccgg tagtgttaga
     2701 cctgaacaag gtttactaaa aatccgtaaa gaacttcaat tgtacgccaa cttaagacca
     2761 tgtaactttg catccgactc tcttttagac ttatctccaa tcaagccaca atttgctaaa
     2821 ggtactgact tcgttgttgt cagagaatta gtgggaggta tttactttgg taagagaaag
     2881 gaggacgatg gtgatggtgt cgcttgggat agtgaacaat acaccgttcc agaagtgcaa
     2941 agaatcacaa gaatggccgc tttcatggcc ctacaacatg agccaccatt gcctatttgg
     3001 tccttggata aagctaatgt tttggcctct tcaagattat ggagaaaaac tgtggaggaa
     3061 accatcaaga acgaatttcc tacattgaag gttcaacatc aattgattga ttctgccgcc
     3121 atgatcctag ttaagaaccc aacccaccta aatggtatta taatcaccag caacatgttt
     3181 ggtgatatca tctccgatga agcctccgtt atcccaggtt ccttgggttt gttgccatct
     3241 gcgtccttgg cctctttgcc agacaagaac accgcatttg gtttgtacga accatgccac
     3301 ggttctgctc cagatttgcc aaagaataag gtcaacccta tcgccactat cttgtctgct
     3361 gcaatgatgt tgaaattgtc attgaacttg cctgaagaag gtaaggccat tgaagatgca
     3421 gttaaaaagg ttttggatgc aggtatcaga actggtgatt taggtggttc caacagtacc
     3481 accgaagtcg gtgatgctgt cgccgaagaa gttaagaaaa tccttgctta acctggagga
     3541 cccttctctt tagactattc tactcttatg cacgtaaaaa attctaggaa atatgtatta
     3601 actaggagta aaataaccgg ctagtggcat tcatatagcc gtctgtttac atctacatca
     3661 cacatttcga gtgtatatct cgcaacgttg gcgttaaata ggcaggagta ctcgtatcgc
     3721 atgtcggtgc gacacgaaat tacaaaatgg aatatgttca tagggtagac gaaactatat
     3781 acgcaatcta catacattta tcaagaagga gaaaaaggag gatgtaaagg aatacaggta
     3841 agcaaattga tactaatggc tcaacgtgat aaggaaaaag aattgcactt taacattaat
     3901 attgacaagg aggagggcac cacacaaaaa gttaggtgta acagaaaatc atgaaactat
     3961 gattcctaat ttatatattg gaggattttc tctaaaaaaa aaaaaataca acaaataaaa
     4021 aacactcaat gacctgacca tttgatggag tttaagtcaa taccttcttg aaccatttcc
     4081 cataatggtg aaagttccct caagaatttt actctgtcag aaacggcctt aacgacgtag
     4141 tcgacctcct cttcagtact aaatctacca ataccaaatc tgatggaaga atgggctaat
     4201 gcatcatcct tacccagcgc cgagcggccg cgtgttacaa ccaattaacc aattctgatt
     4261 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     4321 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     4381 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     4441 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     4501 aatccggtga gaatggcaaa agcttatgca tttctttcca gacttgttca acaggccagc
     4561 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     4621 cctgagcgag gcgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     4681 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     4741 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     4801 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     4861 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     4921 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     4981 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     5041 tggagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     5101 aagcagacag ttttattgtt catgatgata tatttttatc ttgtgcaatg taacatcaga
     5161 gattttgaga cacaacgtgg ctttgttgaa taaatcgaac ttttgctgag ttgaaggatc
     5221 agtcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     5281 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5341 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5401 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     5461 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5521 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5581 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5641 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5701 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5761 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5821 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5881 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     5941 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtgcgg ccgccaatac
     6001 ccagaagaac agtaaaataa agcaaggtac gtgaaattaa tatttttaaa tggttctaac
     6061 cgatgccgaa gaactgcgca gtccggttat aacgtctgac atgtcctttt ttgatttgga
     6121 atccaaccac tcaagtgact ctgttcattt actttgcgaa aaatataccc acaaattgcc
     6181 catcgaaagt gaatcgcaaa ccaccttcag actggcaccg acaaagcaaa gattatacag
     6241 acagagtact ttatacgtac cgttaagtct caagcaaagg gttttcttat ttactgaacg
     6301 ggtaaagagt atctgggccg gcttgccaag atgcaaaccg aataagtatt tcaaagttgc
     6361 atttgcctta gccgtcctga caccattggc tatttggata ttttatattg actttcgtgt
     6421 acattgatca catcgactgt tctattggca aatgaaccac gggcattgac tatttttcag
     6481 gttactacta tatattatca tcacgggcaa ggattgtacc ctgaattcgc atctagatgg
     6541 tagagccaca aacagccggt acaagcaacg atctccagga ccatctgaat catgcgcgga
     6601 tgacacgaac tcacgacggc gatcacagac attaacccac agtacagaca ctgcgacaac
     6661 gtggcaattc gtcgcaatac cgtctcactg aactggccga taattgcaga cgaacgcagt
     6721 tcgagtttat cattatcaat actgccattt caaagaatac gtaaataatt aatagtagtg
     6781 attttcctaa ctttatttag tcaaaaaatt agccttttaa ttctgctgta acccgtacat
     6841 gcccaaaata gggggcgggt tacacagaat atataacatc gtaggtgtct gggtgaacag
     6901 tttattcctg gcatccacta aatataatgg agcccgcttt ttaagctggc atccagaaaa
     6961 aaaaagaatc ccagcaccaa aatattgttt tcttcaccaa ccatcagttc ataggtccat
     7021 tctcttagcg caactacaga gaacaggggc acaaacaggc aaaaaacggg cacaacctca
     7081 atggagtgat gcaacctgcc tggagtaaat gatgacacaa ggcaattgac ccacgcatgt
     7141 atctatctca ttttcttaca ccttctatta ccttctgctc tctctgattt ggaaaaagct
     7201 gaaaaaaaag gttgaaacca gttccctgaa attattcccc tacttgacta ataagtatat
     7261 aaagacggta ggtattgatt gtaattctgt aaatctattt cttaaacttc ttaaattcta
     7321 cttttatagt tagtcttttt tttagtttta aaacaccaag aacttagttt cgaataaaca
     7381 cacataaaca aacaaa
//
