LOCUS       sequence-266837-        6379 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GFP-targeting sgRNAs to the left (G) and right (H) of 
            iCas9 recognition sequence to be used in yeast dual reporter system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6379)
  AUTHORS   Standage-Beier K, Brookhouser N, Balachandran P, Zhang Q, Brafman 
            DA, Wang X
  TITLE     RNA-Guided Recombinase-Cas9 Fusion Targets Genomic DNA Deletion and 
            Integration.
  JOURNAL   CRISPR J. 2019 Aug;2:209-222. doi: 10.1089/crispr.2019.0013.
  PUBMED    31436506
REFERENCE   2  (bases 1 to 6379)
  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: "CRISPR J.";
            date: "2019-08"; volume: "2:209-222. doi"; pages: " 
            10.1089/crispr.2019.0013"
COMMENT     Sequence Label: pRS424-ysg(G:H)
FEATURES             Location/Qualifiers
     source          1..6379
                     /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        187..467
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             468..1142
                     /codon_start=1
                     /gene="S. cerevisiae TRP1"
                     /product="phosphoribosylanthranilate isomerase, required 
                     for tryptophan biosynthesis"
                     /label=TRP1
                     /note="yeast auxotrophic marker"
                     /translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
                     RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
                     WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
                     VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
                     KK"
     rep_origin      complement(1241..1696)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1378..1399)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1590..1609
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1826..1848
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1840..1857
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1841..1857
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1867..1886
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1867..1885
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1910..1926
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1911..1927
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1948..2216
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        2237..2312
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        2337..2605
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        2626..2701
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2777..2797)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2777..2795)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2816..2832)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2816..2832)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2829..2851)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2840..2856
                     /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(2864..2870,2871..2888,2889..2894))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2909..2930
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3047..3064)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3218..3806)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3298..3317)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3977..4768,4769..4837))
                     /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     4600..4619
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4838..4942)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4969..6311
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5932..5979)
                     /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     6352..6370
                     /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 accataaacg acattactat atatataata taggaagcat ttaatagaca gcatcgtaat
      241 atatgtgtac tttgcagtta tgacgccaga tggcagtagt ggaagatatt ctttattgaa
      301 aaatagcttg tcaccttacg tacaatcttg atccggagct tttctttttt tgccgattaa
      361 gaattaattc ggtcgaaaaa agaaaaggag agggccaaga gggagggcat tggtgactat
      421 tgagcacgtg agtatacgtg attaagcaca caaaggcagc ttggagtatg tctgttatta
      481 atttcacagg tagttctggt ccattggtga aagtttgcgg cttgcagagc acagaggccg
      541 cagaatgtgc tctagattcc gatgctgact tgctgggtat tatatgtgtg cccaatagaa
      601 agagaacaat tgacccggtt attgcaagga aaatttcaag tcttgtaaaa gcatataaaa
      661 atagttcagg cactccgaaa tacttggttg gcgtgtttcg taatcaacct aaggaggatg
      721 ttttggctct ggtcaatgat tacggcattg atatcgtcca actgcatgga gatgagtcgt
      781 ggcaagaata ccaagagttc ctcggtttgc cagttattaa aagactcgta tttccaaaag
      841 actgcaacat actactcagt gcagcttcac agaaacctca ttcgtttatt cccttgtttg
      901 attcagaagc aggtgggaca ggtgaacttt tggattggaa ctcgatttct gactgggttg
      961 gaaggcaaga gagccccgaa agcttacatt ttatgttagc tggtggactg acgccagaaa
     1021 atgttggtga tgcgcttaga ttaaatggcg ttattggtgt tgatgtaagc ggaggtgtgg
     1081 agacaaatgg tgtaaaagac tctaacaaaa tagcaaattt cgtcaaaaat gctaagaaat
     1141 aggttattac tgagtagtat ttatttaagt attgtttgtg cacttgccta tgcggtgtga
     1201 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt
     1261 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     1321 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     1381 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     1441 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     1501 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     1561 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     1621 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     1681 ccgctacagg gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     1741 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     1801 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag
     1861 cgcgcgtaat acgactcact atagggcgaa ttgggtaccg ggccccccct cgaggtcgac
     1921 ggtatcgata agcttgatat cgaattctct ttgaaaagat aatgtatgat tatgctttca
     1981 ctcatattta tacagaaact tgatgttttc tttcgagtat atacaaggtg attacatgta
     2041 cgtttgaagt acaactctag attttgtagt gccctcttgg gctagcggta aaggtgcgca
     2101 ttttttcaca ccctacaatg ttctgttcaa aagattttgg tcaaacgctg tagaagtgaa
     2161 agttggtgcg catgtttcgg cgttcgaaac ttctccgcag tgaaagataa atgatcacgt
     2221 acgagtgcaa gcctgggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     2281 atcaacttga aaaagtggca ccgagtcggt gcttttttta ctagataatt ggagactctt
     2341 tgaaaagata atgtatgatt atgctttcac tcatatttat acagaaactt gatgttttct
     2401 ttcgagtata tacaaggtga ttacatgtac gtttgaagta caactctaga ttttgtagtg
     2461 ccctcttggg ctagcggtaa aggtgcgcat tttttcacac cctacaatgt tctgttcaaa
     2521 agattttggt caaacgctgt agaagtgaaa gttggtgcgc atgtttcggc gttcgaaact
     2581 tctccgcagt gaaagataaa tgatcacaga ccatactcca gatgggtttt agagctagaa
     2641 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     2701 ctttttttac tagataattg gagactctac tagttctaga gcggccgcca ccgcggtgga
     2761 gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat
     2821 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     2881 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     2941 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     3001 aacgcgcggg gagaggcggt ttgcgtattg ggcgcgcttc cgcttcctcg ctcactgact
     3061 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3121 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3181 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3241 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3301 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3361 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3421 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3481 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3541 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3601 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     3661 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3721 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3781 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3841 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3901 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3961 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4021 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4081 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4141 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4201 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4261 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     4321 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4381 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4441 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     4501 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     4561 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     4621 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     4681 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4741 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4801 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     4861 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4921 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa cgaagcatct
     4981 gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat
     5041 ctgagctgca tttttacaga acagaaatgc aacgcgaaag cgctatttta ccaacgaaga
     5101 atctgtgctt catttttgta aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa
     5161 agaatctgag ctgcattttt acagaacaga aatgcaacgc gagagcgcta ttttaccaac
     5221 aaagaatcta tacttctttt ttgttctaca aaaatgcatc ccgagagcgc tatttttcta
     5281 acaaagcatc ttagattact ttttttctcc tttgtgcgct ctataatgca gtctcttgat
     5341 aactttttgc actgtaggtc cgttaaggtt agaagaaggc tactttggtg tctattttct
     5401 cttccataaa aaaagcctga ctccacttcc cgcgtttact gattactagc gaagctgcgg
     5461 gtgcattttt tcaagataaa ggcatccccg attatattct ataccgatgt ggattgcgca
     5521 tactttgtga acagaaagtg atagcgttga tgattcttca ttggtcagaa aattatgaac
     5581 ggtttcttct attttgtctc tatatactac gtataggaaa tgtttacatt ttcgtattgt
     5641 tttcgattca ctctatgaat agttcttact acaatttttt tgtctaaaga gtaatactag
     5701 agataaacat aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg
     5761 atgggtaggt tatataggga tatagcacag agatatatag caaagagata cttttgagca
     5821 atgtttgtgg aagcggtatt cgcaatattt tagtagctcg ttacagtccg gtgcgttttt
     5881 ggttttttga aagtgcgtct tcagagcgct tttggttttc aaaagcgctc tgaagttcct
     5941 atactttcta gagaatagga acttcggaat aggaacttca aagcgtttcc gaaaacgagc
     6001 gcttccgaaa atgcaacgcg agctgcgcac atacagctca ctgttcacgt cgcacctata
     6061 tctgcgtgtt gcctgtatat atatatacat gagaagaacg gcatagtgcg tgtttatgct
     6121 taaatgcgta cttatatgcg tctatttatg taggatgaaa ggtagtctag tacctcctgt
     6181 gatattatcc cattccatgc ggggtatcgt atgcttcctt cagcactacc ctttagctgt
     6241 tctatatgct gccactcctc aattggatta gtctcatcct tcaatgctat catttccttt
     6301 gatattggat catattaaga aaccattatt atcatgacat taacctataa aaataggcgt
     6361 atcacgaggc cctttcgtc
//
