LOCUS       sequence-318180-        5648 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Helper plasmid for cloning gRNAs under the control of the SNR52 
            promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5648)
  AUTHORS   Dong C, Jiang L, Xu S, Huang L, Cai J, Lian J, Xu Z
  TITLE     A Single Cas9-VPR Nuclease for Simultaneous Gene Activation, 
            Repression, and Editing in Saccharomyces cerevisiae.
  JOURNAL   ACS Synth Biol. 2020 Sep 18;9(9):2252-2257. doi: 
            10.1021/acssynbio.0c00218. Epub 2020 Aug 28.
  PUBMED    32841560
REFERENCE   2  (bases 1 to 5648)
  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"; doi: 
            "10.1021/acssynbio.0c00218"; journalName: "ACS Synth Biol"; date: 
            "2020-09-18- 18"; volume: "9"; issue: "9"; pages: "2252-2257"
COMMENT     Sequence Label: pRS423-SgH
FEATURES             Location/Qualifiers
     source          1..5648
                     /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        317..503
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             504..1163
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL
                     ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     primer_bind     complement(1560..1581)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1657..1925
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        1942..2017
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2029..2048
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(2086..2102)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2086..2102)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2099..2121)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2110..2126
                     /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(2134..2140,2141..2158,2159..2164))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2179..2200
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2317..2334)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2488..3076)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2568..2587)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3247..4038,4039..4107))
                     /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     3870..3889
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4108..4212)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4239..5581
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5202..5249)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     5621..5639
                     /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 accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt
      241 gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta
      301 ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat
      361 tttttttttt cccctagcgg atgactcttt ttttttctta gcgattggca ttatcacata
      421 atgaattata cattatataa agtaatgtga tttcttcgaa gaatatacta aaaaatgagc
      481 aggcaagata aacgaaggca aagatgacag agcagaaagc cctagtaaag cgtattacaa
      541 atgaaaccaa gattcagatt gcgatctctt taaagggtgg tcccctagcg atagagcact
      601 cgatcttccc agaaaaagag gcagaagcag tagcagaaca ggccacacaa tcgcaagtga
      661 ttaacgtcca cacaggtata gggtttctgg accatatgat acatgctctg gccaagcatt
      721 ccggctggtc gctaatcgtt gagtgcattg gtgacttaca catagacgac catcacacca
      781 ctgaagactg cgggattgct ctcggtcaag cttttaaaga ggccctactg gcgcgtggag
      841 taaaaaggtt tggatcagga tttgcgcctt tggatgaggc actttccaga gcggtggtag
      901 atctttcgaa caggccgtac gcagttgtcg aacttggttt gcaaagggag aaagtaggag
      961 atctctcttg cgagatgatc ccgcattttc ttgaaagctt tgcagaggct agcagaatta
     1021 ccctccacgt tgattgtctg cgaggcaaga atgatcatca ccgtagtgag agtgcgttca
     1081 aggctcttgc ggttgccata agagaagcca cctcgcccaa tggtaccaac gatgttccct
     1141 ccaccaaagg tgttcttatg tagtgacacc gattatttaa agctgcagca tacgatatat
     1201 atacatgtgt atatatgtat acctatgaat gtcagtaagt atgtatacga acagtatgat
     1261 actgaagatg acaaggtaat gcatcattct atacgtgtca ttctgaacga ggcgcgcttt
     1321 ccttttttct ttttgctttt tctttttttt tctcttgaac tcgacggatc tatgcggtgt
     1381 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta aacgttaata
     1441 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     1501 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     1561 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     1621 ccgtctatca gggcgatggc ccactacgtg ctcgagtctt tgaaaagata atgtatgatt
     1681 atgctttcac tcatatttat acagaaactt gatgttttct ttcgagtata tacaaggtga
     1741 ttacatgtac gtttgaagta caactctaga ttttgtagtg ccctcttggg ctagcggtaa
     1801 aggtgcgcat tttttcacac cctacaatgt tctgttcaaa agattttggt caaacgctgt
     1861 agaagtgaaa gttggtgcgc atgtttcggc gttcgaaact tctccgcagt gaaagataaa
     1921 tgatcggaga ccgaggtctc cgttttagag ctagaaatag caagttaaaa taaggctagt
     1981 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgatcctt tttttgtttt
     2041 ttatgtctga gctccctgca ggcatgcaag cttggcgtaa tcatggtcat agctgtttcc
     2101 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     2161 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     2221 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     2281 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     2341 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     2401 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     2461 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     2521 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     2581 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     2641 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     2701 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     2761 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     2821 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     2881 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     2941 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     3001 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     3061 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     3121 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     3181 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     3241 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     3301 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     3361 tggccccagt gctgcaatga taccgcggct cccacgctca ccggctccag atttatcagc
     3421 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     3481 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     3541 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     3601 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     3661 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     3721 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     3781 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     3841 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     3901 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     3961 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     4021 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     4081 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     4141 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     4201 aggggttccg cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt
     4261 ttgtagaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca
     4321 tttttacaga acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt
     4381 catttttgta aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag
     4441 ctgcattttt acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta
     4501 tacttctttt ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc
     4561 ttagattact ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc
     4621 actgtaggtc cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa
     4681 aaaagcctga ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt
     4741 tcaagataaa ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga
     4801 acagaaagtg atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct
     4861 attttgtctc tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca
     4921 ctctatgaat agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat
     4981 aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt
     5041 tatataggga tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg
     5101 aagcggtatt cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga
     5161 aagtgcgtct tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta
     5221 gagaatagga acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa
     5281 atgcaacgcg agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt
     5341 gcctgtatat atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta
     5401 cttatatgcg tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc
     5461 cattccatgc ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct
     5521 gccactcctc aattggatta gtctcatcct tcaatgctat catttccttt gatattggat
     5581 catctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     5641 ctttcgtc
//
