LOCUS       sequence-273639-        5973 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 5973)
  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 5973)
  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..5973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     13..35
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     27..44
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     28..44
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     54..73
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        54..72
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     97..113
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     98..114
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        135..403
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        422..497
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(553..573)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(553..571)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(592..608)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(592..608)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(605..627)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    616..632
                     /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(640..646,647..664,665..670))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    685..706
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(823..840)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(994..1582)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1074..1093)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1753..2544,2545..2613))
                     /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     2376..2395
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2614..2718)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2745..4087
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(3708..3755)
                     /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     4128..4146
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4184..4206)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4306..4325
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4346..4627
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     CDS             4628..5302
                     /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(5405..5860)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5542..5563)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5754..5773
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg
       61 actcactata gggcgaattg ggtaccgggc cccccctcga ggtcgacggt atcgataagc
      121 ttgatatcga attctctttg aaaagataat gtatgattat gctttcactc atatttatac
      181 agaaacttga tgttttcttt cgagtatata caaggtgatt acatgtacgt ttgaagtaca
      241 actctagatt ttgtagtgcc ctcttgggct agcggtaaag gtgcgcattt tttcacaccc
      301 tacaatgttc tgttcaaaag attttggtca aacgctgtag aagtgaaagt tggtgcgcat
      361 gtttcggcgt tcgaaacttc tccgcagtga aagataaatg atcagaagag cgagctcttc
      421 tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
      481 tggcaccgag tcggtgcttt ttttactagt tctagagcgg ccgccaccgc ggtggagctc
      541 cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct
      601 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
      661 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
      721 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
      781 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
      841 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
      901 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
      961 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     1021 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     1081 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     1141 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     1201 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     1261 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     1321 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     1381 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     1441 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     1501 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     1561 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     1621 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     1681 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     1741 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     1801 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     1861 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     1921 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     1981 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     2041 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     2101 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     2161 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     2221 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     2281 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     2341 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     2401 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     2461 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     2521 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     2581 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     2641 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     2701 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgaacgaa gcatctgtgc
     2761 ttcattttgt agaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca aagaatctga
     2821 gctgcatttt tacagaacag aaatgcaacg cgaaagcgct attttaccaa cgaagaatct
     2881 gtgcttcatt tttgtaaaac aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa
     2941 tctgagctgc atttttacag aacagaaatg caacgcgaga gcgctatttt accaacaaag
     3001 aatctatact tcttttttgt tctacaaaaa tgcatcccga gagcgctatt tttctaacaa
     3061 agcatcttag attacttttt ttctcctttg tgcgctctat aatgcagtct cttgataact
     3121 ttttgcactg taggtccgtt aaggttagaa gaaggctact ttggtgtcta ttttctcttc
     3181 cataaaaaaa gcctgactcc acttcccgcg tttactgatt actagcgaag ctgcgggtgc
     3241 attttttcaa gataaaggca tccccgatta tattctatac cgatgtggat tgcgcatact
     3301 ttgtgaacag aaagtgatag cgttgatgat tcttcattgg tcagaaaatt atgaacggtt
     3361 tcttctattt tgtctctata tactacgtat aggaaatgtt tacattttcg tattgttttc
     3421 gattcactct atgaatagtt cttactacaa tttttttgtc taaagagtaa tactagagat
     3481 aaacataaaa aatgtagagg tcgagtttag atgcaagttc aaggagcgaa aggtggatgg
     3541 gtaggttata tagggatata gcacagagat atatagcaaa gagatacttt tgagcaatgt
     3601 ttgtggaagc ggtattcgca atattttagt agctcgttac agtccggtgc gtttttggtt
     3661 ttttgaaagt gcgtcttcag agcgcttttg gttttcaaaa gcgctctgaa gttcctatac
     3721 tttctagaga ataggaactt cggaatagga acttcaaagc gtttccgaaa acgagcgctt
     3781 ccgaaaatgc aacgcgagct gcgcacatac agctcactgt tcacgtcgca cctatatctg
     3841 cgtgttgcct gtatatatat atacatgaga agaacggcat agtgcgtgtt tatgcttaaa
     3901 tgcgtactta tatgcgtcta tttatgtagg atgaaaggta gtctagtacc tcctgtgata
     3961 ttatcccatt ccatgcgggg tatcgtatgc ttccttcagc actacccttt agctgttcta
     4021 tatgctgcca ctcctcaatt ggattagtct catccttcaa tgctatcatt tcctttgata
     4081 ttggatcata ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     4141 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     4201 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     4261 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     4321 ttgtactgag agtgcaccat agatcaacga cattactata tatataatat aggaagcatt
     4381 taatagaaca gcatcgtaat atatgtgtac tttgcagtta tgacgccaga tggcagtagt
     4441 ggaagatatt ctttattgaa aaatagcttg tcaccttacg tacaatcttg atccggagct
     4501 tttctttttt tgccgattaa gaattaattc ggtcgaaaaa agaaaaggag agggccaaga
     4561 gggagggcat tggtgactat tgagcacgtg agtatacgtg attaagcaca caaaggcagc
     4621 ttggagtatg tctgttatta atttcacagg tagttctggt ccattggtga aagtttgcgg
     4681 cttgcagagc acagaggccg cagaatgtgc tctagattcc gatgctgact tgctgggtat
     4741 tatatgtgtg cccaatagaa agagaacaat tgacccggtt attgcaagga aaatttcaag
     4801 tcttgtaaaa gcatataaaa atagttcagg cactccgaaa tacttggttg gcgtgtttcg
     4861 taatcaacct aaggaggatg ttttggctct ggtcaatgat tacggcattg atatcgtcca
     4921 actgcatgga gatgagtcgt ggcaagaata ccaagagttc ctcggtttgc cagttattaa
     4981 aagactcgta tttccaaaag actgcaacat actactcagt gcagcttcac agaaacctca
     5041 ttcgtttatt cccttgtttg attcagaagc aggtgggaca ggtgaacttt tggattggaa
     5101 ctcgatttct gactgggttg gaaggcaaga gagccccgaa agcttacatt ttatgttagc
     5161 tggtggactg acgccagaaa atgttggtga tgcgcttaga ttaaatggcg ttattggtgt
     5221 tgatgtaagc ggaggtgtgg agacaaatgg tgtaaaagac tctaacaaaa tagcaaattt
     5281 cgtcaaaaat gctaagaaat aggttattac tgagtagtat ttatttaagt attgtttgtg
     5341 cacttgccga tctatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     5401 caggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     5461 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     5521 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     5581 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     5641 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     5701 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     5761 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     5821 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc
     5881 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     5941 ggggatgtgc tgcaaggcga ttaagttggg taa
//
