LOCUS       sequence-66634-e        5202 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast CEN/ARS vector (Ura3) that contains sgRNA controlled by SNR 52
            promoter, targeting endogenous TRE elements of pTET07 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5202)
  AUTHORS   Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, 
            Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, 
            Weissman JS, Qi LS
  TITLE     CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in 
            Eukaryotes.
  JOURNAL   Cell. 2013 Jul 9. pii: S0092-8674(13)00826-X. doi: 
            10.1016/j.cell.2013.06.044.
  PUBMED    23849981
REFERENCE   2  (bases 1 to 5202)
  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: "Cell. 2013 
            Jul 9. pii: S0092-8674(13)00826-X. doi: 10.1016/j.cell.2013.06.044."
COMMENT     Sequence Label: pSNR52-sgTET
FEATURES             Location/Qualifiers
     source          1..5202
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..52
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     44..61
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     45..61
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     68..87
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        68..86
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        112..380
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        401..475
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      480..499
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        complement(528..546)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(567..583)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(567..583)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(580..602)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    591..607
                     /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(615..621,622..639,640..645))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    660..681
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(798..815)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(969..1557)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(1728..2519,2520..2588))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2351..2370
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2589..2693)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    2730..3233
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     3275..3293
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3331..3353)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3453..3472
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3497..3712
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             3713..4516
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(4647..5102)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4784..4805)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4996..5015
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
       61 tgaattgtaa tacgactcac tatagggcga attggagctc actcggaatt ctctttgaaa
      121 agataatgta tgattatgct ttcactcata tttatacaga aacttgatgt tttctttcga
      181 gtatatacaa ggtgattaca tgtacgtttg aagtacaact ctagattttg tagtgccctc
      241 ttgggctagc ggtaaaggtg cgcatttttt cacaccctac aatgttctgt tcaaaagatt
      301 ttggtcaaac gctgtagaag tgaaagttgg tgcgcatgtt tcggcgttcg aaacttctcc
      361 gcagtgaaag ataaatgatc acttttctct atcactgata gttttagagc tagaaatagc
      421 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtggtgct
      481 ttttttgttt tttatgtctc tgcagagttc ggtaccagct tttgttccct ttagtgaggg
      541 ttaatttcga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
      601 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
      661 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
      721 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg ttttcgtatt
      781 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
      841 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
      901 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
      961 ctggcgtttt tccataggct cggcccccct gacgagcatc acaaaaatcg acgctcaagt
     1021 cagaggtggc gaaacccgac aggactataa agataccagg cgttcccccc tggaagctcc
     1081 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     1141 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
     1201 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     1261 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     1321 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     1381 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     1441 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     1501 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     1561 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     1621 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     1681 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     1741 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactg
     1801 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     1861 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     1921 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     1981 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     2041 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     2101 caacgatcaa ggcgagttac atgatccccc atgttgtgaa aaaaagcggt tagctccttc
     2161 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     2221 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     2281 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     2341 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     2401 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     2461 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     2521 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     2581 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     2641 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     2701 ccccgaaaag tgccacctgg gtccttttca tcacgtgcta taaaaataat tataatttaa
     2761 attttttaat ataaatatat aaattaaaaa tagaaagtaa aaaaagaaat taaagaaaaa
     2821 atagtttttg ttttccgaag atgtaaaaga ctctaggggg atcgccaaca aatactacct
     2881 tttatcttgc tcttcctgct ctcaggtatt aatgccgaat tgtttcatct tgtctgtgta
     2941 gaagaccaca cacgaaaatc ctgtgatttt acattttact tatcgttaat cgaatgtata
     3001 tctatttaat ctgcttttct tgtctaataa atatatatgt aaagtacgct ttttgttgaa
     3061 attttttaaa cctttgttta tttttttttc ttcattccgt aactcttcta ccttctttat
     3121 ttactttcta aaatccaaat acaaaacata aaaataaata aacacagagt aaattcccaa
     3181 attattccat cattaaaaga tacgaggcgc gtgtaagtta caggcaagcg atccgtccta
     3241 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     3301 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     3361 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     3421 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     3481 gcaccacgct tttcaattca attcatcatt ttttttttat tctttttttt gatttcggtt
     3541 tccttgaaat ttttttgatt cggtaatctc cgaacagaag gaagaacgaa ggaaggagca
     3601 cagacttaga ttggtatata tacgcatatg tagtgttgaa gaaacatgaa attgcccagt
     3661 attcttaacc caactgcaca gaacaaaaac gtgcaggaaa cgaagataaa tcatgtcgaa
     3721 agctacatat aaggaacgtg ctgctactca tcctagtcct gttgctgcca agctatttaa
     3781 tatcatgcac gaaaagcaaa caaacttgtg tgcttcattg gatgttcgta ccaccaagga
     3841 attactggag ttagttgaag cattaggtcc caaaatttgt ttactaaaaa cacatgtgga
     3901 tatcttgact gatttttcca tggagggcac agttaagccg ctaaaggcat tatccgccaa
     3961 gtacaatttt ttactcttcg aagacagaaa atttgctgac attggtaata cagtcaaatt
     4021 gcagtactct gcgggtgtat acagaatagc agaatgggca gacattacga atgcacacgg
     4081 tgtggtgggc ccaggtattg ttagcggttt gaagcaggcg gcagaagaag taacaaagga
     4141 acctagaggc cttttgatgt tagcagaatt gtcatgcaag ggctccctat ctactggaga
     4201 atatactaag ggtactgttg acattgcgaa gagcgacaaa gattttgtta tcggctttat
     4261 tgctcaaaga gacatgggtg gaagagatga aggttacgat tggttgatta tgacacccgg
     4321 tgtgggttta gatgacaagg gagacgcatt gggtcaacag tatagaaccg tggatgatgt
     4381 ggtctctaca ggatctgaca ttattattgt tggaagagga ctatttgcaa agggaaggga
     4441 tgctaaggta gagggtgaac gttacagaaa agcaggctgg gaagcatatt tgagaagatg
     4501 cggccagcaa aactaaaaaa ctgtattata agtaaatgca tgtatactaa actcacaaat
     4561 tagagcttca atttaattat atcagttatt accctatgcg gtgtgaaata ccgcacagat
     4621 gcgtaaggag aaaataccgc atcaggaaat tgtaaacgtt aatattttgt taaaattcgc
     4681 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     4741 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     4801 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     4861 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     4921 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     4981 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     5041 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     5101 gtcgcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     5161 ttcgctatta cgccagctgg cgaagggggg atgtgctgca ag
//
