LOCUS       sequence-323625-        5653 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 5653)
  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 5653)
  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..5653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     26..44
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(82..104)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     204..223
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        370..557
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             558..1220
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     primer_bind     complement(1617..1638)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1714..1982
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        1999..2074
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2086..2105
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(2143..2159)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2143..2159)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2156..2178)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2167..2183
                     /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(2191..2197,2198..2215,2216..2221))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2236..2257
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2374..2391)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2545..3133)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2625..2644)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3304..4095,4096..4164))
                     /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     3927..3946
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4165..4269)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4296..5638
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5259..5306)
                     /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)."
ORIGIN
        1 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
       61 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
      121 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
      181 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccataa
      241 attcccgttt taagagcttg gtgagcgcta ggagtcactg ccaggtatcg tttgaacacg
      301 gcattagtca gggaagtcat aacacagtcc tttcccgcaa ttttcttttt ctattactct
      361 tggcctcctc tagtacactc tatatttttt tatgcctcgg taatgatttt catttttttt
      421 tttccaccta gcggatgact cttttttttt cttagcgatt ggcattatca cataatgaat
      481 tatacattat ataaagtaat gtgatttctt cgaagaatat actaaaaaat gagcaggcaa
      541 gataaacgaa ggcaaagatg acagagcaga aagccctagt aaagcgtatt acaaatgaaa
      601 ccaagattca gattgcgatc tctttaaagg gtggtcccct agcgatagag cactcgatct
      661 tcccagaaaa agaggcagaa gcagtagcag aacaggccac acaatcgcaa gtgattaacg
      721 tccacacagg tatagggttt ctggaccata tgatacatgc tctggccaag cattccggct
      781 ggtcgctaat cgttgagtgc attggtgact tacacataga cgaccatcac accactgaag
      841 actgcgggat tgctctcggt caagctttta aagaggccct aggggccgtg cgtggagtaa
      901 aaaggtttgg atcaggattt gcgcctttgg atgaggcact ttccagagcg gtggtagatc
      961 tttcgaacag gccgtacgca gttgtcgaac ttggtttgca aagggagaaa gtaggagatc
     1021 tctcttgcga gatgatcccg cattttcttg aaagctttgc agaggctagc agaattaccc
     1081 tccacgttga ttgtctgcga ggcaagaatg atcatcaccg tagtgagagt gcgttcaagg
     1141 ctcttgcggt tgccataaga gaagccacct cgcccaatgg taccaacgat gttccctcca
     1201 ccaaaggtgt tcttatgtag tgacaccgat tatttaaagc tgcagcatac gatatatata
     1261 catgtgtata tatgtatacc tatgaatgtc agtaagtatg tatacgaaca gtatgatact
     1321 gaagatgaca aggtaatgca tcattctata cgtgtcattc tgaacgaggc gcgctttcct
     1381 tttttctttt tgctttttct ttttttttct cttgaactcg acggatctat gcggtgtgaa
     1441 ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc gttaatattt
     1501 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     1561 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     1621 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     1681 tctatcaggg cgatggccca ctacgtgctc gagtctttga aaagataatg tatgattatg
     1741 ctttcactca tatttataca gaaacttgat gttttctttc gagtatatac aaggtgatta
     1801 catgtacgtt tgaagtacaa ctctagattt tgtagtgccc tcttgggcta gcggtaaagg
     1861 tgcgcatttt ttcacaccct acaatgttct gttcaaaaga ttttggtcaa acgctgtaga
     1921 agtgaaagtt ggtgcgcatg tttcggcgtt cgaaacttct ccgcagtgaa agataaatga
     1981 tcggagaccg aggtctccgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     2041 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt gatccttttt ttgtttttta
     2101 tgtctgagct ccctgcaggc atgcaagctt ggcgtaatca tggtcatagc tgtttcctgt
     2161 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     2221 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     2281 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     2341 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     2401 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     2461 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     2521 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     2581 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     2641 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     2701 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     2761 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     2821 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     2881 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     2941 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     3001 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     3061 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3121 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3181 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     3241 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     3301 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     3361 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     3421 ccccagtgct gcaatgatac cgcggctccc acgctcaccg gctccagatt tatcagcaat
     3481 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3541 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     3601 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     3661 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     3721 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     3781 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     3841 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     3901 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     3961 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     4021 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     4081 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     4141 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     4201 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     4261 ggttccgcgc acatttcccc gaaaagtgcc acctgaacga agcatctgtg cttcattttg
     4321 tagaacaaaa atgcaacgcg agagcgctaa tttttcaaac aaagaatctg agctgcattt
     4381 ttacagaaca gaaatgcaac gcgaaagcgc tattttacca acgaagaatc tgtgcttcat
     4441 ttttgtaaaa caaaaatgca acgcgagagc gctaattttt caaacaaaga atctgagctg
     4501 catttttaca gaacagaaat gcaacgcgag agcgctattt taccaacaaa gaatctatac
     4561 ttcttttttg ttctacaaaa atgcatcccg agagcgctat ttttctaaca aagcatctta
     4621 gattactttt tttctccttt gtgcgctcta taatgcagtc tcttgataac tttttgcact
     4681 gtaggtccgt taaggttaga agaaggctac tttggtgtct attttctctt ccataaaaaa
     4741 agcctgactc cacttcccgc gtttactgat tactagcgaa gctgcgggtg cattttttca
     4801 agataaaggc atccccgatt atattctata ccgatgtgga ttgcgcatac tttgtgaaca
     4861 gaaagtgata gcgttgatga ttcttcattg gtcagaaaat tatgaacggt ttcttctatt
     4921 ttgtctctat atactacgta taggaaatgt ttacattttc gtattgtttt cgattcactc
     4981 tatgaatagt tcttactaca atttttttgt ctaaagagta atactagaga taaacataaa
     5041 aaatgtagag gtcgagttta gatgcaagtt caaggagcga aaggtggatg ggtaggttat
     5101 atagggatat agcacagaga tatatagcaa agagatactt ttgagcaatg tttgtggaag
     5161 cggtattcgc aatattttag tagctcgtta cagtccggtg cgtttttggt tttttgaaag
     5221 tgcgtcttca gagcgctttt ggttttcaaa agcgctctga agttcctata ctttctagag
     5281 aataggaact tcggaatagg aacttcaaag cgtttccgaa aacgagcgct tccgaaaatg
     5341 caacgcgagc tgcgcacata cagctcactg ttcacgtcgc acctatatct gcgtgttgcc
     5401 tgtatatata tatacatgag aagaacggca tagtgcgtgt ttatgcttaa atgcgtactt
     5461 atatgcgtct atttatgtag gatgaaaggt agtctagtac ctcctgtgat attatcccat
     5521 tccatgcggg gtatcgtatg cttccttcag cactaccctt tagctgttct atatgctgcc
     5581 actcctcaat tggattagtc tcatccttca atgctatcat ttcctttgat attggatcat
     5641 actaagaaac cat
//
