LOCUS       sequence-266838-        5968 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a non-targeting sgRNA to be used as a control in a 
            yeast dual reporter system.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5968)
  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 5968)
  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(NT)
FEATURES             Location/Qualifiers
     source          1..5968
                     /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        2235..2310
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2366..2386)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2366..2384)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2405..2421)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2405..2421)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2418..2440)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2429..2445
                     /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(2453..2459,2460..2477,2478..2483))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2498..2519
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2636..2653)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2807..3395)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2887..2906)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3566..4357,4358..4426))
                     /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     4189..4208
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4427..4531)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4558..5900
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5521..5568)
                     /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     5941..5959
                     /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 atgatcagaa
     2221 gagcgagctc ttctgtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     2281 caacttgaaa aagtggcacc gagtcggtgc tttttttact agttctagag cggccgccac
     2341 cgcggtggag ctccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat
     2401 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     2461 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     2521 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     2581 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgcgcttcc gcttcctcgc
     2641 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     2701 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     2761 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     2821 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     2881 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     2941 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3001 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3061 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3121 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3181 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3241 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3301 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3361 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3421 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3481 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     3541 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     3601 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     3661 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     3721 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     3781 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     3841 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     3901 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     3961 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4021 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4081 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4141 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     4201 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     4261 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     4321 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4381 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4441 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4501 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgaac
     4561 gaagcatctg tgcttcattt tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa
     4621 acaaagaatc tgagctgcat ttttacagaa cagaaatgca acgcgaaagc gctattttac
     4681 caacgaagaa tctgtgcttc atttttgtaa aacaaaaatg caacgcgaga gcgctaattt
     4741 ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg agagcgctat
     4801 tttaccaaca aagaatctat acttcttttt tgttctacaa aaatgcatcc cgagagcgct
     4861 atttttctaa caaagcatct tagattactt tttttctcct ttgtgcgctc tataatgcag
     4921 tctcttgata actttttgca ctgtaggtcc gttaaggtta gaagaaggct actttggtgt
     4981 ctattttctc ttccataaaa aaagcctgac tccacttccc gcgtttactg attactagcg
     5041 aagctgcggg tgcatttttt caagataaag gcatccccga ttatattcta taccgatgtg
     5101 gattgcgcat actttgtgaa cagaaagtga tagcgttgat gattcttcat tggtcagaaa
     5161 attatgaacg gtttcttcta ttttgtctct atatactacg tataggaaat gtttacattt
     5221 tcgtattgtt ttcgattcac tctatgaata gttcttacta caattttttt gtctaaagag
     5281 taatactaga gataaacata aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc
     5341 gaaaggtgga tgggtaggtt atatagggat atagcacaga gatatatagc aaagagatac
     5401 ttttgagcaa tgtttgtgga agcggtattc gcaatatttt agtagctcgt tacagtccgg
     5461 tgcgtttttg gttttttgaa agtgcgtctt cagagcgctt ttggttttca aaagcgctct
     5521 gaagttccta tactttctag agaataggaa cttcggaata ggaacttcaa agcgtttccg
     5581 aaaacgagcg cttccgaaaa tgcaacgcga gctgcgcaca tacagctcac tgttcacgtc
     5641 gcacctatat ctgcgtgttg cctgtatata tatatacatg agaagaacgg catagtgcgt
     5701 gtttatgctt aaatgcgtac ttatatgcgt ctatttatgt aggatgaaag gtagtctagt
     5761 acctcctgtg atattatccc attccatgcg gggtatcgta tgcttccttc agcactaccc
     5821 tttagctgtt ctatatgctg ccactcctca attggattag tctcatcctt caatgctatc
     5881 atttcctttg atattggatc atattaagaa accattatta tcatgacatt aacctataaa
     5941 aataggcgta tcacgaggcc ctttcgtc
//
