LOCUS       sequence-52745-e        8776 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses VP16-CIB1 for light-inducible protein expression when 
            coupled with GalBD-CRY2PHR or GalBD-CRY2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8776)
  AUTHORS   Hughes RM, Bolger S, Tapadia H, Tucker CL
  TITLE     Light-mediated control of DNA transcription in yeast.
  JOURNAL   Methods. 2012 Aug 15.
  PUBMED    22922268
REFERENCE   2  (bases 1 to 8776)
  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: "Methods. 
            2012 Aug 15."
COMMENT     Sequence Label: pRMH124-VP16-CIB1
FEATURES             Location/Qualifiers
     source          1..8776
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        771..1476
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             1498..1518
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1519..1752
                     /codon_start=1
                     /gene="UL48"
                     /product="transcriptional activation domain of herpes 
                     simplex virus protein VP16 (Triezenberg et al., 1988; 
                     Cousens et al., 1989)"
                     /label=VP16 AD
                     /translation="APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGF
                     TPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     CDS             1801..2310
                     /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      3204..3391
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3259..3283)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(3508..4602)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(4603..5008)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(5050..5066)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5050..5066)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5063..5085)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5074..5090
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5098..5104,5105..5122,5123..5128))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5143..5164
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    5219..5252
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5330..5363
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5432..5449)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5603..6191)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5683..5702)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6362..7153,7154..7222))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6985..7004
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7223..7327)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7395..7413
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7451..7473)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7573..7592
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      7609..8773
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 tgcatgcctg caggtcgaga tccgggatcg aagaaatgat ggtaaatgaa ataggaaatc
       61 aaggagcatg aaggcaaaag acaaatataa gggtcgaacg aaaaataaag tgaaaagtgt
      121 tgatatgatg tatttggctt tgcggcgccg aaaaaacgag tttacgcaat tgcacaatca
      181 tgctgactct gtggcggacc cgcgctcttg ccggcccggc gataacgctg ggcgtgaggc
      241 tgtgcccggc ggagtttttt gcgcctgcat tttccaaggt ttaccctgcg ctaaggggcg
      301 agattggaga agcaataaga atgccggttg gggttgcgat gatgacgacc acgacaactg
      361 gtgtcattat ttaagttgcc gaaagaacct gagtgcattt gcaacatgag tatactagaa
      421 gaatgagcca agacttgcga gacgcgagtt tgccggtggt gcgaacaata gagcgaccat
      481 gaccttgaag gtgagacgcg cataaccgct agagtacttt gaagaggaaa cagcaatagg
      541 gttgctacca gtataaatag acaggtacat acaacactgg aaatggttgt ctgtttgagt
      601 acgctttcaa ttcatttggg tgtgcacttt attatgttac aatatggaag ggaactttac
      661 acttctccta tgcacatata ttaattaaag tccaatgcta gtagagaagg ggggtaacac
      721 ccctccgcgc tcttttccga tttttttcta aaccgtggaa tatttcggat atccttttgt
      781 tgtttccggg tgtacaatat ggacttcctc ttttctggca accaaaccca tacatcggga
      841 ttcctataat accttcgttg gtctccctaa catgtaggtg gcggagggga gatatacaat
      901 agaacagata ccagacaaga cataatgggc taaacaagac tacaccaatt acactgcctc
      961 attgatggtg gtacataacg aactaatact gtagccctag acttgatagc catcatcata
     1021 tcgaagtttc actacccttt ttccatttgc catctattga agtaataata ggcgcatgca
     1081 acttcttttc tttttttttc ttttctctct cccccgttgt tgtctcacca tatccgcaat
     1141 gacaaaaaaa atgatggaag acactaaagg aaaaaattaa cgacaaagac agcaccaaca
     1201 gatgtcgttg ttccagagct gatgaggggt atctcgaagc acacgaaact ttttccttcc
     1261 ttcattcacg cacactactc tctaatgagc aacggtatac ggccttcctt ccagttactt
     1321 gaatttgaaa taaaaaaaag tttgctgtct tgctatcaag tataaataga cctgcaatta
     1381 ttaatctttt gtttcctcgt cattgttctc gttccctttc ttccttgttt ctttttctgc
     1441 acaatatttc aagctatacc aagcatacaa tcaactccaa gctttgcaaa gatgggccct
     1501 aaaaagaagc gtaaagtcgc ccccccgacc gatgtcagcc tgggggacga gctccactta
     1561 gacggcgagg acgtggcgat ggcgcatgcc gacgcgctag acgatttcga tctggacatg
     1621 ttgggggacg gggattcccc gggtccggga tttacccccc acgactccgc cccctacggc
     1681 gctctggata tggccgactt cgagtttgag cagatgttta ccgatgccct tggaattgac
     1741 gagtacggtg gggaattccc ggggatccgt cgacgcgtct gcagaagctt cggcggcagc
     1801 atgaatggag ctataggagg tgaccttttg ctcaattttc ctgacatgtc ggtcctagag
     1861 cgccaaaggg ctcacctcaa gtacctcaat cccacctttg attctcctct cgccggcttc
     1921 tttgccgatt cttcaatgat taccggcggc gagatggaca gctatctttc gactgccggt
     1981 ttgaatcttc cgatgatgta cggtgagacg acggtggaag gtgattcaag actctcaatt
     2041 tcgccggaaa cgacgcttgg gactggaaat ttcaagaaac ggaagtttga tacagagact
     2101 aaggattgta atgagaagaa gaagaagatg acgatgaaca gagatgacct agtagaagaa
     2161 ggagaagaag agaagtcgaa aataacagag caaaacaatg ggagcacaaa aagcatcaag
     2221 aagatgaaac acaaagccaa gaaagaagag aacaatttct ctaatgattc atctaaagtg
     2281 acgaaggaat tggagaaaac ggattatatt catgttcgtg cacgacgagg ccaagccact
     2341 gatagtcaca gcatagcaga acgagttaga agagaaaaga tcagtgagag aatgaagttt
     2401 ctacaagatt tggttcctgg atgcgacaag atcacaggca aagcagggat gcttgatgaa
     2461 atcattaact atgttcagtc tcttcagaga caaatcgagt tcttatcgat gaaactagca
     2521 attgtgaatc caaggccgga ttttgatatg gatgacattt ttgccaaaga ggttgcctca
     2581 actccaatga ctgtggtgcc atctcctgaa atggttcttt ccggttattc tcatgagatg
     2641 gttcactctg gttattctag tgagatggtt aactccggtt accttcatgt caatccaatg
     2701 cagcaagtga ataccagttc tgatccattg tcatgcttca acaatggcga agctccttcg
     2761 atgtgggact ctcatgtgca gaatctctat ggcaatttag gagtaccggt catcgagctc
     2821 gagctgcaga tgaatcgtag atactgaaaa accccgcaag ttcacttcaa ctgtgcatcg
     2881 tgcaccatct caatttcttt catttataca tcgttttgcc ttcttttatg taactatact
     2941 cctctaagtt tcaatcttgg ccatgtaacc tctgatctat agaatttttt aaatgactag
     3001 aattaatgcc catctttttt ttggacctaa attcttcatg aaaatatatt acgagggctt
     3061 attcagaagc tttggacttc ttcgccagag gtttggtcaa gtctccaatc aaggttgtcg
     3121 gcttgtctac cttgccagaa atttacgaaa agatggaaaa gggtcaaatc gttggtagat
     3181 acgttgttga cacttctaaa taagcgaatt tcttatgatt tatgattttt attattaaat
     3241 aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa
     3301 aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagcatg
     3361 aggtcgctct tattgaccac acctctaccg gccggtcgaa attcccctac cctatgaaca
     3421 tattccattt tgtaatttcg tgtcgtttct attatgaatt tcatttataa agtttatgta
     3481 caaatatcat aaaaaaagag aatcttttta agcaaggatt ttcttaactt cttcggcgac
     3541 agcatcaccg acttcggtgg tactgttgga accacctaaa tcaccagttc tgatacctgc
     3601 atccaaaacc tttttaactg catcttcaat ggccttacct tcttcaggca agttcaatga
     3661 caatttcaac atcattgcag cagacaagat agtggcgata gggttgacct tattctttgg
     3721 caaatctgga gcagaaccgt ggcatggttc gtacaaacca aatgcggtgt tcttgtctgg
     3781 caaagaggcc aaggacgcag atggcaacaa acccaaggaa cctgggataa cggaggcttc
     3841 atcggagatg atatcaccaa acatgttgct ggtgattata ataccattta ggtgggttgg
     3901 gttcttaact aggatcatgg cggcagaatc aatcaattga tgttgaacct tcaatgtagg
     3961 aaattcgttc ttgatggttt cctccacagt ttttctccat aatcttgaag aggccaaaac
     4021 attagcttta tccaaggacc aaataggcaa tggtggctca tgttgtaggg ccatgaaagc
     4081 ggccattctt gtgattcttt gcacttctgg aacggtgtat tgttcactat cccaagcgac
     4141 accatcacca tcgtcttcct ttctcttacc aaagtaaata cctcccacta attctctgac
     4201 aacaacgaag tcagtacctt tagcaaattg tggcttgatt ggagataagt ctaaaagaga
     4261 gtcggatgca aagttacatg gtcttaagtt ggcgtacaat tgaagttctt tacggatttt
     4321 tagtaaacct tgttcaggtc taacactacc tgtaccccat ttaggaccac ccacagcacc
     4381 taacaaaacg gcatcaacct tcttggaggc ttccagcgcc tcatctggaa gtgggacacc
     4441 tgtagcgtcg atagcagcac caccaattaa atgattttcg aaatcgaact tgacattgga
     4501 acgaacatca gaaatagctt taagaacctt aatggcttcg gctgtgattt cttgaccaac
     4561 gtggtcacct ggcaaaacga cgatcttctt aggggcagac attagaatgg tatatccttg
     4621 aaatatatat atatattgct gaaatgtaaa aggtaagaaa agttagaaag taagacgatt
     4681 gctaaccacc tattggaaaa aacaataggt ccttaaataa tattgtcaac ttcaagtatt
     4741 gtgatgcaag catttagtca tgaacgcttc tctattctat atgaaaagcc ggttccggcg
     4801 ctctcacctt tcctttttct cccaattttt cagttgaaaa aggtatatgc gtcaggcgac
     4861 ctctgaaatt aacaaaaaat ttccagtcat cgaatttgat tctgtgcgat agcgcccctg
     4921 tgtgttctcg ttatgttgag gaaaaaaata atggttgcta agagattcga actcttgcat
     4981 cttacgatac ctgagtattc ccacagttgg ggatctcgac tctagctaga ggatcaattc
     5041 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     5101 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     5161 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgat
     5221 aacttcgtat aatgtatgct atacgaagtt attaggtctg aagaggagtt tacgtccagc
     5281 caagctagct tggctgcagg tcgagcggcc gcgatccgga acccttaata taacttcgta
     5341 taatgtatgc tatacgaagt tatcagctgc attaatgaat cggccaacgc gcggggagag
     5401 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5461 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5521 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5581 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5641 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5701 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5761 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5821 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5881 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5941 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     6001 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     6061 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6121 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6181 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6241 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6301 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6361 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6421 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6481 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6541 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6601 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6661 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6721 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6781 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6841 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6901 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6961 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     7021 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     7081 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     7141 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     7201 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     7261 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     7321 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     7381 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     7441 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     7501 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     7561 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccataac gcatttaagc
     7621 ataaacacgc actatgccgt tcttctcatg tatatatata tacaggcaac acgcagatat
     7681 aggtgcgacg tgaacagtga gctgtatgtg cgcagctcgc gttgcatttt cggaagcgct
     7741 cgttttcgga aacgctttga agttcctatt ccgaagttcc tattctctag ctagaaagta
     7801 taggaacttc agagcgcttt tgaaaaccaa aagcgctctg aagacgcact ttcaaaaaac
     7861 caaaaacgca ccggactgta acgagctact aaaatattgc gaataccgct tccacaaaca
     7921 ttgctcaaaa gtatctcttt gctatatatc tctgtgctat atccctatat aacctaccca
     7981 tccacctttc gctccttgaa cttgcatcta aactcgacct ctacattttt tatgtttatc
     8041 tctagtatta ctctttagac aaaaaaattg tagtaagaac tattcataga gtgaatcgaa
     8101 aacaatacga aaatgtaaac atttcctata cgtagtatat agagacaaaa tagaagaaac
     8161 cgttcataat tttctgacca atgaagaatc atcaacgcta tcactttctg ttcacaaagt
     8221 atgcgcaatc cacatcggta tagaatataa tcggggatgc ctttatcttg aaaaaatgca
     8281 cccgcagctt cgctagtaat cagtaaacgc gggaagtgga gtcaggcttt ttttatggaa
     8341 gagaaaatag acaccaaagt agccttcttc taaccttaac ggacctacag tgcaaaaagt
     8401 tatcaagaga ctgcattata gagcgcacaa aggagaaaaa aagtaatcta agatgctttg
     8461 ttagaaaaat agcgctctcg ggatgcattt ttgtagaaca aaaaagaagt atagattctt
     8521 tgttggtaaa atagcgctct cgcgttgcat ttctgttctg taaaaatgca gctcagattc
     8581 tttgtttgaa aaattagcgc tctcgcgttg catttttgtt ttacaaaaat gaagcacaga
     8641 ttcttcgttg gtaaaatagc gctttcgcgt tgcatttctg ttctgtaaaa atgcagctca
     8701 gattctttgt ttgaaaaatt agcgctctcg cgttgcattt ttgttctaca aaatgaagca
     8761 cagatgcttc gttgct
//
