LOCUS       sequence-323627-        5587 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Helper plasmid for cloning gRNAs under the control of the tRNA(Pro) 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5587)
  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 5587)
  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-ProSgH
FEATURES             Location/Qualifiers
     source          1..5587
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(125..172)
                     /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     545..563
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(601..623)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     723..742
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        889..1076
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             1077..1739
                     /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(2136..2157)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_RNA        2452..2527
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2539..2558
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(2596..2612)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2596..2612)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2609..2631)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2620..2636
                     /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(2644..2650,2651..2668,2669..2674))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2689..2710
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2827..2844)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2998..3586)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3078..3097)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3757..4548,4549..4617))
                     /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     4380..4399
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4618..4722)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(4749..5587,1..504)
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
ORIGIN
        1 atacttttga gcaatgtttg tggaagcggt attcgcaata ttttagtagc tcgttacagt
       61 ccggtgcgtt tttggttttt tgaaagtgcg tcttcagagc gcttttggtt ttcaaaagcg
      121 ctctgaagtt cctatacttt ctagagaata ggaacttcgg aataggaact tcaaagcgtt
      181 tccgaaaacg agcgcttccg aaaatgcaac gcgagctgcg cacatacagc tcactgttca
      241 cgtcgcacct atatctgcgt gttgcctgta tatatatata catgagaaga acggcatagt
      301 gcgtgtttat gcttaaatgc gtacttatat gcgtctattt atgtaggatg aaaggtagtc
      361 tagtacctcc tgtgatatta tcccattcca tgcggggtat cgtatgcttc cttcagcact
      421 accctttagc tgttctatat gctgccactc ctcaattgga ttagtctcat ccttcaatgc
      481 tatcatttcc tttgatattg gatcatacta agaaaccatt attatcatga cattaaccta
      541 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
      601 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
      661 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
      721 tgcggcatca gagcagattg tactgagagt gcaccataaa ttcccgtttt aagagcttgg
      781 tgagcgctag gagtcactgc caggtatcgt ttgaacacgg cattagtcag ggaagtcata
      841 acacagtcct ttcccgcaat tttctttttc tattactctt ggcctcctct agtacactct
      901 atattttttt atgcctcggt aatgattttc attttttttt ttccacctag cggatgactc
      961 tttttttttc ttagcgattg gcattatcac ataatgaatt atacattata taaagtaatg
     1021 tgatttcttc gaagaatata ctaaaaaatg agcaggcaag ataaacgaag gcaaagatga
     1081 cagagcagaa agccctagta aagcgtatta caaatgaaac caagattcag attgcgatct
     1141 ctttaaaggg tggtccccta gcgatagagc actcgatctt cccagaaaaa gaggcagaag
     1201 cagtagcaga acaggccaca caatcgcaag tgattaacgt ccacacaggt atagggtttc
     1261 tggaccatat gatacatgct ctggccaagc attccggctg gtcgctaatc gttgagtgca
     1321 ttggtgactt acacatagac gaccatcaca ccactgaaga ctgcgggatt gctctcggtc
     1381 aagcttttaa agaggcccta ggggccgtgc gtggagtaaa aaggtttgga tcaggatttg
     1441 cgcctttgga tgaggcactt tccagagcgg tggtagatct ttcgaacagg ccgtacgcag
     1501 ttgtcgaact tggtttgcaa agggagaaag taggagatct ctcttgcgag atgatcccgc
     1561 attttcttga aagctttgca gaggctagca gaattaccct ccacgttgat tgtctgcgag
     1621 gcaagaatga tcatcaccgt agtgagagtg cgttcaaggc tcttgcggtt gccataagag
     1681 aagccacctc gcccaatggt accaacgatg ttccctccac caaaggtgtt cttatgtagt
     1741 gacaccgatt atttaaagct gcagcatacg atatatatac atgtgtatat atgtatacct
     1801 atgaatgtca gtaagtatgt atacgaacag tatgatactg aagatgacaa ggtaatgcat
     1861 cattctatac gtgtcattct gaacgaggcg cgctttcctt ttttcttttt gctttttctt
     1921 tttttttctc ttgaactcga cggatctatg cggtgtgaaa taccgcacag atgcgtaagg
     1981 agaaaatacc gcatcaggaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     2041 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     2101 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     2161 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     2221 tacgtgctcg agtccgtagt ctaaatgagt tacgtacgaa agggagccaa aagcaatctg
     2281 accaatttgt atatatatac atctatccga aaaggaagac atcaattagt acgggcgtgt
     2341 ggtctagtgg tatgattctc gctttgggcg acttcctgat taaacaggaa gacaaagcat
     2401 gcgagaggcc ctgggttcaa ttcccagctc gccccggaga ccgaggtctc cgttttagag
     2461 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
     2521 tcggtgcttt tttgatcctt tttttgtttt ttatgtctga gctccctgca ggcatgcaag
     2581 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     2641 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     2701 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     2761 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     2821 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     2881 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     2941 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3001 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3061 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3121 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3181 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3241 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3301 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3361 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3421 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3481 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3541 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3601 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3661 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3721 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     3781 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     3841 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcggct
     3901 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     3961 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4021 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4081 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     4141 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     4201 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4261 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4321 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4381 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4441 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     4501 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     4561 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     4621 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     4681 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     4741 gccacctgaa cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc
     4801 taatttttca aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgaaag
     4861 cgctatttta ccaacgaaga atctgtgctt catttttgta aaacaaaaat gcaacgcgag
     4921 agcgctaatt tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc
     4981 gagagcgcta ttttaccaac aaagaatcta tacttctttt ttgttctaca aaaatgcatc
     5041 ccgagagcgc tatttttcta acaaagcatc ttagattact ttttttctcc tttgtgcgct
     5101 ctataatgca gtctcttgat aactttttgc actgtaggtc cgttaaggtt agaagaaggc
     5161 tactttggtg tctattttct cttccataaa aaaagcctga ctccacttcc cgcgtttact
     5221 gattactagc gaagctgcgg gtgcattttt tcaagataaa ggcatccccg attatattct
     5281 ataccgatgt ggattgcgca tactttgtga acagaaagtg atagcgttga tgattcttca
     5341 ttggtcagaa aattatgaac ggtttcttct attttgtctc tatatactac gtataggaaa
     5401 tgtttacatt ttcgtattgt tttcgattca ctctatgaat agttcttact acaatttttt
     5461 tgtctaaaga gtaatactag agataaacat aaaaaatgta gaggtcgagt ttagatgcaa
     5521 gttcaaggag cgaaaggtgg atgggtaggt tatataggga tatagcacag agatatatag
     5581 caaagag
//
