LOCUS       sequence-273641-        6384 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 6384)
  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 6384)
  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..6384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        37..112
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(188..208)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(188..206)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(227..243)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(227..243)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(240..262)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    251..267
                     /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(275..281,282..299,300..305))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    320..341
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(458..475)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(629..1217)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(709..728)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1388..2179,2180..2248))
                     /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     2011..2030
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2249..2353)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2380..3722
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(3343..3390)
                     /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     3763..3781
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3819..3841)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3941..3960
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3981..4262
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             4263..4937
                     /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(5040..5495)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5177..5198)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5389..5408
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5621..5643
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5635..5652
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5636..5652
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5662..5681
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5662..5680
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     5705..5721
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     5706..5722
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        5743..6011
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        6032..6107
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        join(6132..6384,1..16)
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
ORIGIN
        1 tgaaagataa atgatcacag accatactcc agatgggttt tagagctaga aatagcaagt
       61 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcttttttta
      121 ctagataatt ggagactcta ctagttctag agcggccgcc accgcggtgg agctccagct
      181 tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc
      241 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
      301 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
      361 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
      421 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
      481 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
      541 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
      601 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
      661 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
      721 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
      781 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
      841 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
      901 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
      961 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     1021 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     1081 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     1141 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     1201 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     1261 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     1321 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     1381 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     1441 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     1501 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     1561 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     1621 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     1681 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     1741 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     1801 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     1861 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     1921 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     1981 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     2041 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     2101 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     2161 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     2221 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     2281 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     2341 taggggttcc gcgcacattt ccccgaaaag tgccacctga acgaagcatc tgtgcttcat
     2401 tttgtagaac aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc
     2461 atttttacag aacagaaatg caacgcgaaa gcgctatttt accaacgaag aatctgtgct
     2521 tcatttttgt aaaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca aagaatctga
     2581 gctgcatttt tacagaacag aaatgcaacg cgagagcgct attttaccaa caaagaatct
     2641 atacttcttt tttgttctac aaaaatgcat cccgagagcg ctatttttct aacaaagcat
     2701 cttagattac tttttttctc ctttgtgcgc tctataatgc agtctcttga taactttttg
     2761 cactgtaggt ccgttaaggt tagaagaagg ctactttggt gtctattttc tcttccataa
     2821 aaaaagcctg actccacttc ccgcgtttac tgattactag cgaagctgcg ggtgcatttt
     2881 ttcaagataa aggcatcccc gattatattc tataccgatg tggattgcgc atactttgtg
     2941 aacagaaagt gatagcgttg atgattcttc attggtcaga aaattatgaa cggtttcttc
     3001 tattttgtct ctatatacta cgtataggaa atgtttacat tttcgtattg ttttcgattc
     3061 actctatgaa tagttcttac tacaattttt ttgtctaaag agtaatacta gagataaaca
     3121 taaaaaatgt agaggtcgag tttagatgca agttcaagga gcgaaaggtg gatgggtagg
     3181 ttatataggg atatagcaca gagatatata gcaaagagat acttttgagc aatgtttgtg
     3241 gaagcggtat tcgcaatatt ttagtagctc gttacagtcc ggtgcgtttt tggttttttg
     3301 aaagtgcgtc ttcagagcgc ttttggtttt caaaagcgct ctgaagttcc tatactttct
     3361 agagaatagg aacttcggaa taggaacttc aaagcgtttc cgaaaacgag cgcttccgaa
     3421 aatgcaacgc gagctgcgca catacagctc actgttcacg tcgcacctat atctgcgtgt
     3481 tgcctgtata tatatataca tgagaagaac ggcatagtgc gtgtttatgc ttaaatgcgt
     3541 acttatatgc gtctatttat gtaggatgaa aggtagtcta gtacctcctg tgatattatc
     3601 ccattccatg cggggtatcg tatgcttcct tcagcactac cctttagctg ttctatatgc
     3661 tgccactcct caattggatt agtctcatcc ttcaatgcta tcatttcctt tgatattgga
     3721 tcatactaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     3781 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     3841 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     3901 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
     3961 ctgagagtgc accatagatc aacgacatta ctatatatat aatataggaa gcatttaata
     4021 gaacagcatc gtaatatatg tgtactttgc agttatgacg ccagatggca gtagtggaag
     4081 atattcttta ttgaaaaata gcttgtcacc ttacgtacaa tcttgatccg gagcttttct
     4141 ttttttgccg attaagaatt aattcggtcg aaaaaagaaa aggagagggc caagagggag
     4201 ggcattggtg actattgagc acgtgagtat acgtgattaa gcacacaaag gcagcttgga
     4261 gtatgtctgt tattaatttc acaggtagtt ctggtccatt ggtgaaagtt tgcggcttgc
     4321 agagcacaga ggccgcagaa tgtgctctag attccgatgc tgacttgctg ggtattatat
     4381 gtgtgcccaa tagaaagaga acaattgacc cggttattgc aaggaaaatt tcaagtcttg
     4441 taaaagcata taaaaatagt tcaggcactc cgaaatactt ggttggcgtg tttcgtaatc
     4501 aacctaagga ggatgttttg gctctggtca atgattacgg cattgatatc gtccaactgc
     4561 atggagatga gtcgtggcaa gaataccaag agttcctcgg tttgccagtt attaaaagac
     4621 tcgtatttcc aaaagactgc aacatactac tcagtgcagc ttcacagaaa cctcattcgt
     4681 ttattccctt gtttgattca gaagcaggtg ggacaggtga acttttggat tggaactcga
     4741 tttctgactg ggttggaagg caagagagcc ccgaaagctt acattttatg ttagctggtg
     4801 gactgacgcc agaaaatgtt ggtgatgcgc ttagattaaa tggcgttatt ggtgttgatg
     4861 taagcggagg tgtggagaca aatggtgtaa aagactctaa caaaatagca aatttcgtca
     4921 aaaatgctaa gaaataggtt attactgagt agtatttatt taagtattgt ttgtgcactt
     4981 gccgatctat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga
     5041 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     5101 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     5161 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     5221 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     5281 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     5341 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     5401 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     5461 acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac
     5521 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     5581 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     5641 acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccgggccc
     5701 cccctcgagg tcgacggtat cgataagctt gatatcgaat tctctttgaa aagataatgt
     5761 atgattatgc tttcactcat atttatacag aaacttgatg ttttctttcg agtatataca
     5821 aggtgattac atgtacgttt gaagtacaac tctagatttt gtagtgccct cttgggctag
     5881 cggtaaaggt gcgcattttt tcacacccta caatgttctg ttcaaaagat tttggtcaaa
     5941 cgctgtagaa gtgaaagttg gtgcgcatgt ttcggcgttc gaaacttctc cgcagtgaaa
     6001 gataaatgat cacgtacgag tgcaagcctg ggttttagag ctagaaatag caagttaaaa
     6061 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttactaga
     6121 taattggaga ctctttgaaa agataatgta tgattatgct ttcactcata tttatacaga
     6181 aacttgatgt tttctttcga gtatatacaa ggtgattaca tgtacgtttg aagtacaact
     6241 ctagattttg tagtgccctc ttgggctagc ggtaaaggtg cgcatttttt cacaccctac
     6301 aatgttctgt tcaaaagatt ttggtcaaac gctgtagaag tgaaagttgg tgcgcatgtt
     6361 tcggcgttcg aaacttctcc gcag
//
