LOCUS       sequence-331070-        6251 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for expression of sgRNAs in yeast: SNR52p-NotI-sgRNA-SUP4t 
            (NotI site in place of the spacer).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6251)
  AUTHORS   Goldberg GW, Spencer JM, Giganti DO, Camellato BR, Agmon N, Ichikawa
            DM, Boeke JD, Noyes MB
  TITLE     Engineered dual selection for directed evolution of SpCas9 PAM 
            specificity.
  JOURNAL   Nat Commun. 2021 Jan 13;12(1):349. doi: 10.1038/s41467-020-20650-x.
  PUBMED    33441553
REFERENCE   2  (bases 1 to 6251)
  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: "Nat 
            Commun."; date: "2021-01-13"; volume: "12(1):349. doi"; pages: " 
            10.1038/s41467-020-20650-x"
COMMENT     Sequence Label: pNA0304
FEATURES             Location/Qualifiers
     source          1..6251
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     112..131
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     365..382
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    499..520
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(535..540,541..558,559..565)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    573..589
                     /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)."
     primer_bind     578..600
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     597..613
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     597..613
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     632..652
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        634..652
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        670..938
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        947..1021
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1026..1045
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      1051..1298
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1316..1335)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1317..1335)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1345..1362)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1345..1361)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1354..1376)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1502..1957
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1589..1608)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1799..1820
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2088..2891)
                     /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"
     promoter        complement(2892..3107)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(3131..3150)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3250..3272
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3310..3328)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      3369..4711
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    3701..3748
                     /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)."
     promoter        4738..4842
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4843..4911,4912..5703)
                     /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     complement(5061..5080)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5874..6251,1..211)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
       61 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
      121 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
      181 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
      241 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
      301 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
      361 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
      421 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
      481 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
      541 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
      601 aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa
      661 gctggagctt ctttgaaaag ataatgtatg attatgcttt cactcatatt tatacagaaa
      721 cttgatgttt tctttcgagt atatacaagg tgattacatg tacgtttgaa gtacaactct
      781 agattttgta gtgccctctt gggctagcgg taaaggtgcg cattttttca caccctacaa
      841 tgttctgttc aaaagatttt ggtcaaacgc tgtagaagtg aaagttggtg cgcatgtttc
      901 ggcgttcgaa acttctccgc agtgaaagat aaatgatcgc ggccgcgttt tagagctaga
      961 aatagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
     1021 ggtgcttttt ttgtttttta tgtcttcgag tcatgtaatt agttatgtca cgcttacgtt
     1081 cacgccctcc ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct
     1141 aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa
     1201 tttttctttt ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc
     1261 ttgagaaggt tttgggacgc tcgaaggctt taatttgcgg ccggtaccca attcgcccta
     1321 tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa
     1381 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     1441 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     1501 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     1561 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     1621 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     1681 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     1741 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     1801 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     1861 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     1921 aacgcgaatt ttaacaaaat attaacgctt acaatttcct gatgcggtat tttctcctta
     1981 cgcatctgtg cggtatttca caccgcatag ggtaataact gatataatta aattgaagct
     2041 ctaatttgtg agtttagtat acatgcattt acttataata cagtttttta gttttgctgg
     2101 ccgcatcttc tcaaatatgc ttcccagcct gcttttctgt aacgttcacc ctctacctta
     2161 gcatcccttc cctttgcaaa tagtcctctt ccaacaataa taatgtcaga tcctgtagag
     2221 accacatcat ccacggttct atactgttga cccaatgcgt ctcccttgtc atctaaaccc
     2281 acaccgggtg tcataatcaa ccaatcgtaa ccttcatctc ttccacccat gtctctttga
     2341 gcaataaagc cgataacaaa atctttgtcg ctcttcgcaa tgtcaacagt acccttagta
     2401 tattctccag tagataggga gcccttgcat gacaattctg ctaacatcaa aaggcctcta
     2461 ggttcctttg ttacttcttc tgccgcctgc ttcaaaccgc taacaatacc tgggcccacc
     2521 acaccgtgtg cattcgtaat gtctgcccat tctgctattc tgtatacacc cgcagagtac
     2581 tgcaatttga ctgtattacc aatgtcagca aattttctgt cttcgaagag taaaaaattg
     2641 tacttggcgg ataatgcctt tagcggctta actgtgccct ccatggaaaa atcagtcaag
     2701 atatccacat gtgtttttag taaacaaatt ttgggaccta atgcttcaac taactccagt
     2761 aattccttgg tggtacgaac atccaatgaa gcacacaagt ttgtttgctt ttcgtgcatg
     2821 atattaaata gcttggcagc aacaggacta ggatgagtag cagcacgttc cttatatgta
     2881 gctttcgaca tgatttatct tcgtttcctg caggtttttg ttctgtgcag ttgggttaag
     2941 aatactgggc aatttcatgt ttcttcaaca ctacatatgc gtatatatac caatctaagt
     3001 ctgtgctcct tccttcgttc ttccttctgt tcggagatta ccgaatcaaa aaaatttcaa
     3061 ggaaaccgaa atcaaaaaaa agaataaaaa aaaaatgatg aattgaaaag gtggtatggt
     3121 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     3181 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3241 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3301 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     3361 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg
     3421 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg
     3481 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac
     3541 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata
     3601 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc
     3661 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt
     3721 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa
     3781 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga
     3841 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat
     3901 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct
     3961 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta
     4021 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag
     4081 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc
     4141 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct
     4201 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt
     4261 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg
     4321 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa
     4381 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa
     4441 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta
     4501 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt
     4561 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt
     4621 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt
     4681 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc
     4741 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     4801 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     4861 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     4921 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     4981 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5041 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5101 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5161 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     5221 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     5281 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     5341 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     5401 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     5461 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     5521 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     5581 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     5641 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     5701 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     5761 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     5821 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     5881 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     5941 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6001 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6061 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6121 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6181 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6241 gaactgagat a
//
