LOCUS       sequence-323626-        5564 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Helper plasmid for cloning gRNAs under the control of the tRNA(Tyr) 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5564)
  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 5564)
  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-TyrSgH
FEATURES             Location/Qualifiers
     source          1..5564
                     /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..504
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     CDS             505..1167
                     /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(1564..1585)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_RNA        1857..1932
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1944..1963
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(2001..2017)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2001..2017)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2014..2036)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2025..2041
                     /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(2049..2055,2056..2073,2074..2079))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2094..2115
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2232..2249)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2403..2991)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2483..2502)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3162..3953,3954..4022))
                     /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     3785..3804
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4023..4127)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4154..5496
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5117..5164)
                     /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     5537..5555
                     /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 ccacctagcg gatgactctt tttttttctt agcgattggc attatcacat
      421 aatgaattat acattatata aagtaatgtg atttcttcga agaatatact aaaaaatgag
      481 caggcaagat aaacgaaggc aaagatgaca gagcagaaag ccctagtaaa gcgtattaca
      541 aatgaaacca agattcagat tgcgatctct ttaaagggtg gtcccctagc gatagagcac
      601 tcgatcttcc cagaaaaaga ggcagaagca gtagcagaac aggccacaca atcgcaagtg
      661 attaacgtcc acacaggtat agggtttctg gaccatatga tacatgctct ggccaagcat
      721 tccggctggt cgctaatcgt tgagtgcatt ggtgacttac acatagacga ccatcacacc
      781 actgaagact gcgggattgc tctcggtcaa gcttttaaag aggccctagg ggccgtgcgt
      841 ggagtaaaaa ggtttggatc aggatttgcg cctttggatg aggcactttc cagagcggtg
      901 gtagatcttt cgaacaggcc gtacgcagtt gtcgaacttg gtttgcaaag ggagaaagta
      961 ggagatctct cttgcgagat gatcccgcat tttcttgaaa gctttgcaga ggctagcaga
     1021 attaccctcc acgttgattg tctgcgaggc aagaatgatc atcaccgtag tgagagtgcg
     1081 ttcaaggctc ttgcggttgc cataagagaa gccacctcgc ccaatggtac caacgatgtt
     1141 ccctccacca aaggtgttct tatgtagtga caccgattat ttaaagctgc agcatacgat
     1201 atatatacat gtgtatatat gtatacctat gaatgtcagt aagtatgtat acgaacagta
     1261 tgatactgaa gatgacaagg taatgcatca ttctatacgt gtcattctga acgaggcgcg
     1321 ctttcctttt ttctttttgc tttttctttt tttttctctt gaactcgacg gatctatgcg
     1381 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt
     1441 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     1501 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     1561 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     1621 aaaaccgtct atcagggcga tggcccacta cgtgctcgag aaccatcttg gaaggaccgg
     1681 ataattattt gaaatctctt tttcaattgt atatgtgtta tgtagtatac tctttcttca
     1741 acaattaaat actctcggta gccaagttgg tttaaggcgc aagactgtaa tttatcacta
     1801 cgaaatcttg agatcgggcg ttcgactcgc ccccgggaga ggagaccgag gtctccgttt
     1861 tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca
     1921 ccgagtcggt gcttttttga tccttttttt gttttttatg tctgagctcc ctgcaggcat
     1981 gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     2041 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     2101 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     2161 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     2221 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     2281 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2341 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2401 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2461 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2521 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2581 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2641 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2701 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2761 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2821 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2881 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2941 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3001 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3061 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3121 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3181 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3241 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3301 cggctcccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3361 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3421 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3481 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3541 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3601 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3661 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3721 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3781 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3841 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3901 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3961 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4021 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4081 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4141 aaagtgccac ctgaacgaag catctgtgct tcattttgta gaacaaaaat gcaacgcgag
     4201 agcgctaatt tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc
     4261 gaaagcgcta ttttaccaac gaagaatctg tgcttcattt ttgtaaaaca aaaatgcaac
     4321 gcgagagcgc taatttttca aacaaagaat ctgagctgca tttttacaga acagaaatgc
     4381 aacgcgagag cgctatttta ccaacaaaga atctatactt cttttttgtt ctacaaaaat
     4441 gcatcccgag agcgctattt ttctaacaaa gcatcttaga ttactttttt tctcctttgt
     4501 gcgctctata atgcagtctc ttgataactt tttgcactgt aggtccgtta aggttagaag
     4561 aaggctactt tggtgtctat tttctcttcc ataaaaaaag cctgactcca cttcccgcgt
     4621 ttactgatta ctagcgaagc tgcgggtgca ttttttcaag ataaaggcat ccccgattat
     4681 attctatacc gatgtggatt gcgcatactt tgtgaacaga aagtgatagc gttgatgatt
     4741 cttcattggt cagaaaatta tgaacggttt cttctatttt gtctctatat actacgtata
     4801 ggaaatgttt acattttcgt attgttttcg attcactcta tgaatagttc ttactacaat
     4861 ttttttgtct aaagagtaat actagagata aacataaaaa atgtagaggt cgagtttaga
     4921 tgcaagttca aggagcgaaa ggtggatggg taggttatat agggatatag cacagagata
     4981 tatagcaaag agatactttt gagcaatgtt tgtggaagcg gtattcgcaa tattttagta
     5041 gctcgttaca gtccggtgcg tttttggttt tttgaaagtg cgtcttcaga gcgcttttgg
     5101 ttttcaaaag cgctctgaag ttcctatact ttctagagaa taggaacttc ggaataggaa
     5161 cttcaaagcg tttccgaaaa cgagcgcttc cgaaaatgca acgcgagctg cgcacataca
     5221 gctcactgtt cacgtcgcac ctatatctgc gtgttgcctg tatatatata tacatgagaa
     5281 gaacggcata gtgcgtgttt atgcttaaat gcgtacttat atgcgtctat ttatgtagga
     5341 tgaaaggtag tctagtacct cctgtgatat tatcccattc catgcggggt atcgtatgct
     5401 tccttcagca ctacccttta gctgttctat atgctgccac tcctcaattg gattagtctc
     5461 atccttcaat gctatcattt cctttgatat tggatcatac taagaaacca ttattatcat
     5521 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtc
//
