LOCUS       sequence-108129-        5487 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  encodes one a1 operator site upstream of minimal pCYC1 promoter 
            driving yeast enhanced BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5487)
  AUTHORS   Farzadfard F, Perli SD, Lu TK
  TITLE     Tunable and Multifunctional Eukaryotic Transcription Factors Based 
            on CRISPR/Cas.
  JOURNAL   ACS Synth Biol. 2013 Sep 11.
  PUBMED    23977949
REFERENCE   2  (bases 1 to 5487)
  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: "ACS Synth 
            Biol. 2013 Sep 11."
COMMENT     Sequence Label: 1x_a1gRNAOp_pCYC1m_yeBFP2
FEATURES             Location/Qualifiers
     source          1..5487
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        196..416
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             417..1220
                     /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"
     rep_origin      complement(1351..1806)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1488..1509)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1700..1719
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1936..1958
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1950..1967
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1951..1967
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1977..1996
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1977..1995
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2020..2036
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2021..2037
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      3000..3189
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3228..3248)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3228..3246)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3267..3283)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3267..3283)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3280..3302)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3291..3307
                     /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(3315..3321,3322..3339,3340..3345))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3498..3515)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3669..4257)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3749..3768)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4725..5103)
                     /label=ISS
                     /note="immunostimulatory sequence from the AmpR gene; 
                     contains unmethylated CpG dinucleotides in the context of 
                     5'-AACGTT-3' (Sato et al., 1996)"
     primer_bind     5050..5069
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5288..5392)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5460..5478
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc
      241 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg
      301 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc
      361 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt
      421 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat
      481 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca
      541 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg
      601 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg
      661 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca
      721 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac
      781 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa
      841 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg
      901 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct
      961 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac
     1021 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg
     1081 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa
     1141 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa
     1201 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac
     1261 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac
     1321 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat
     1381 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     1441 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     1501 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     1561 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     1621 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     1681 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     1741 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     1801 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     1861 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     1921 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat
     1981 acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac ggtatcgata
     2041 agcttgatat cgaattcctg cagcccgggt actgtatgta catacagtag gatcctatgg
     2101 catgcatgtg ctctgtatgt atataaaact cttgttttct tcttttctct aaatattctt
     2161 tccttataca ttaggacctt tgcagcataa attactatac ttctatagac acacaaacac
     2221 aaatacacac actaatctag ataaaaaatg gtttccaagg gtgaagaatt attcaccggt
     2281 gttgtcccta ttttggtcga attggacggt gatgtcaacg gtcacaagtt ctccgttaga
     2341 ggtgaaggtg aaggtgacgc taccaacggt aagttgacct tgaagttcat ctgtaccact
     2401 ggtaagttgc cagttccttg gccaactttg gttaccactt tgtctcacgg tgttcaatgt
     2461 ttcgcccgtt acccagatca catgaagcaa catgatttct ttaaatccgc catgccagaa
     2521 ggttacgttc aagaaagaac tatcttcttc aaggacgatg gtacttacaa aactagagcc
     2581 gaagtcaagt tcgaaggtga tactttagtc aatagaatcg aattgaaggg tgttgatttc
     2641 aaggaagatg gtaacatttt gggtcacaag ttagaataca atttcaactc ccataatatt
     2701 tacattatgg ctgttaagca aaagaacggt attaaggtca actttaaaat cagacacaac
     2761 gttgaagacg gttctgtcca attagctgac cactaccaac aaaacactcc aattggtgac
     2821 ggtccagtct tattaccaga ttctcattac ttgtctaccc aatctgtctt gtctaaggac
     2881 ccaaatgaaa agagagatca catggtcttg ttagaatttc gtactgctgc tggtatcact
     2941 ttgggtatgg acgaattgta caagtaactc gagtaagctt ggtaccgcgg ctagctaaga
     3001 tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt
     3061 tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg
     3121 tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga
     3181 cgctcgaaga tccggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg
     3241 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     3301 ctcacaattc cacacaacat aggagccgga agcataaagt gtaaagcctg gggtgcctaa
     3361 tgagtgaggt aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     3421 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     3481 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3541 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3601 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     3661 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     3721 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     3781 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     3841 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     3901 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     3961 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4021 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4081 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     4141 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4201 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4261 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4321 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4381 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4441 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4501 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4561 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4621 agggcgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4681 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4741 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4801 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4861 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4921 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4981 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5041 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5101 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5161 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5221 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5281 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5341 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5401 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     5461 ataggcgtat cacgaggccc tttcgtc
//
