LOCUS       sequence-341519-        8123 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Yeast plasmid encoding a 2-kb gRNA-placeholder flanked by 
            BsmBI/Esp3I restriction sites to facilitate gRNA insertion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8123)
  AUTHORS   Schubert OT, Bloom JS, Sadhu MJ, Kruglyak L
  TITLE     Genome-wide base editor screen identifies regulators of protein 
            abundance in yeast.
  JOURNAL   Elife. 2022 Nov 3;11. pii: 79525. doi: 10.7554/eLife.79525.
  PUBMED    36326816
REFERENCE   2  (bases 1 to 8123)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2022-11-3"; volume: "11. pii: 79525. doi"; pages: " 
            10.7554/eLife.79525"
COMMENT     Sequence Label: pgRNA-backbone
FEATURES             Location/Qualifiers
     source          1..8123
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..62)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      103..1445
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    435..482
                     /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        1472..1576
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1577..1645,1646..2437)
                     /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(1795..1814)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2608..3196
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3097..3116
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3350..3367
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3484..3505
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3520..3525,3526..3543,3544..3550)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3558..3574
                     /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     3563..3585
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3582..3598
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3582..3598
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3617..3637
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3619..3637
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3655..3923
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        5804..5878
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      5883..5902
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     terminator      5908..6155
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(6173..6192)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6174..6192)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6202..6219)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6202..6218)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6211..6233)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6359..6814
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6446..6465)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6656..6677
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6945..7748)
                     /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(7749..7964)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7988..8007)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     join(8107..8123,1..6)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 cagaggtttt caccgtcatc accgaaacgc gccagacgaa agggcctcgt gatacgccta
       61 tttttatagg ttaatgtcat gataataatg gtttcttagt atgatccaat atcaaaggaa
      121 atgatagcat tgaaggatga gactaatcca attgaggagt ggcagcatat agaacagcta
      181 aagggtagtg ctgaaggaag catacgatac cccgcatgga atgggataat atcacaggag
      241 gtactagact acctttcatc ctacataaat agacgcatat aagtacgcat ttaagcataa
      301 acacgcacta tgccgttctt ctcatgtata tatatataca ggcaacacgc agatataggt
      361 gcgacgtgaa cagtgagctg tatgtgcgca gctcgcgttg cattttcgga agcgctcgtt
      421 ttcggaaacg ctttgaagtt cctattccga agttcctatt ctctagaaag tataggaact
      481 tcagagcgct tttgaaaacc aaaagcgctc tgaagacgca ctttcaaaaa accaaaaacg
      541 caccggactg taacgagcta ctaaaatatt gcgaataccg cttccacaaa cattgctcaa
      601 aagtatctct ttgctatata tctctgtgct atatccctat ataacctacc catccacctt
      661 tcgctccttg aacttgcatc taaactcgac ctctacattt tttatgttta tctctagtat
      721 tactctttag acaaaaaaat tgtagtaaga actattcata gagtgaatcg aaaacaatac
      781 gaaaatgtaa acatttccta tacgtagtat atagagacaa aatagaagaa accgttcata
      841 attttctgac caatgaagaa tcatcaacgc tatcactttc tgttcacaaa gtatgcgcaa
      901 tccacatcgg tatagaatat aatcggggat gcctttatct tgaaaaaatg cacccgcagc
      961 ttcgctagta atcagtaaac gcgggaagtg gagtcaggct ttttttatgg aagagaaaat
     1021 agacaccaaa gtagccttct tctaacctta acggacctac agtgcaaaaa gttatcaaga
     1081 gactgcatta tagagcgcac aaaggagaaa aaaagtaatc taagatgctt tgttagaaaa
     1141 atagcgctct cgggatgcat ttttgtagaa caaaaaagaa gtatagattc tttgttggta
     1201 aaatagcgct ctcgcgttgc atttctgttc tgtaaaaatg cagctcagat tctttgtttg
     1261 aaaaattagc gctctcgcgt tgcatttttg ttttacaaaa atgaagcaca gattcttcgt
     1321 tggtaaaata gcgctttcgc gttgcatttc tgttctgtaa aaatgcagct cagattcttt
     1381 gtttgaaaaa ttagcgctct cgcgttgcat ttttgttcta caaaatgaag cacagatgct
     1441 tcgttcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     1501 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     1561 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     1621 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     1681 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     1741 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     1801 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     1861 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     1921 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     1981 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     2041 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     2101 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     2161 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     2221 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     2281 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     2341 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     2401 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     2461 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     2521 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     2581 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     2641 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     2701 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     2761 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     2821 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     2881 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     2941 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3001 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3061 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3121 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3181 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3241 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3301 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     3361 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     3421 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     3481 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     3541 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     3601 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agcttctttg
     3661 aaaagataat gtatgattat gctttcactc atatttatac agaaacttga tgttttcttt
     3721 cgagtatata caaggtgatt acatgtacgt ttgaagtaca actctagatt ttgtagtgcc
     3781 ctcttgggct agcggtaaag gtgcgcattt tttcacaccc tacaatgttc tgttcaaaag
     3841 attttggtca aacgctgtag aagtgaaagt tggtgcgcat gtttcggcgt tcgaaacttc
     3901 tccgcagtga aagataaatg atcgagacgg ttgtaaatga gcacacaaaa tacacatgct
     3961 aaaatattat attctatgac ctttataaaa tcaaccaaaa tcttcttttt aataacttta
     4021 gtatcaataa ttagaatttt tatgttcctt tttgcaaact tttaataaaa atgagcaaaa
     4081 taaaaaaacg ctagttttag taactcgcgt tgttttcttc acctttaata atagctactc
     4141 caccacttgt tcctaagcgg tcagctcctg cttcaatcat tttttgagca tcttcaaatg
     4201 ttctaactcc accagctgct ttaactaaag cattgtcttt aacaactgac ttcattagtt
     4261 taacatcttc aaatgttgca cctgattttg aaaatcctgt tgatgtttta acaaattcta
     4321 atccagcttc aacagctatt tcacaagctt tcatgatttc ttcttttgtt aataaacaat
     4381 tttccataat acatttaaca acatgtgatc cagctgcttt ttttacagct ttcatgtctt
     4441 ctaaaactaa ttcataattt ttgtctttta atgcaccaat atttaatacc atatcaattt
     4501 ctgttgcacc atctttaatt gcttcagaaa cttcgaatgc ttttgtagct gttgtgcatg
     4561 cacctagagg aaaacctaca acatttgtta ttcctacatt tgtgcctttt aataattctt
     4621 tacaatagct tgttcaatat gaattaacac aaactgttgc aaaatcaaat tcaattgctt
     4681 catcacataa ttgtttaatt tcagctttcg tagcatcttg ttttaataat gtgtgatcta
     4741 tatatttgtt tagtttcatt ttttctccta tatattcatt tttaatttta attctttaat
     4801 aatttcgtct actttaactt tagcgttttg aacagattca ccaacaccta taaaataaat
     4861 ttttagttta ggttcagttc cacttgggcg aacagcaaat catgacttat cttctaaata
     4921 aaattttagt aagtcttgtc ctggcatatt atacattcca tcgatgtagt cttcaacatt
     4981 aacaacttta agtccagcaa tttgagttaa gggtgttgct ctcaatgatt tcattaatgg
     5041 ttcaattttt aatttctttt cttctggttt aaaattcaag tttaaagtga aagtgtaata
     5101 tgcacccatt tctttaaata aatcttctaa atagtctact aatgttttat tttgtttttt
     5161 ataaaatcaa gcagcctctg ctattaatat agaagcttgt attccatctt tatctctagc
     5221 tgagtcatca attacatatc cataactttc ttcataagca aaaacaaaat ttaatccgtt
     5281 atcttcttct ttagcaattt ctctacccat tcatttaaat ccagttaaag tttttacaat
     5341 attaactcca tatttttcat gagcgattct atcacccaaa tcacttgtta caaaacttga
     5401 atatagagcc ggattttttg gaatgctatt taagcgtttt agatttgata attttcaatc
     5461 aattaaaatt ggtcctgttt gatttccatc taatcttaca aaatgaccat catgttttat
     5521 tgccattcca aatctgtcag catctgggtc attcataata ataatatctg catcatgttt
     5581 aataccatat tcaagcggta tttttcatgc aggatcaaat tctggatttg gatttacaac
     5641 atttttaaat gtttcatctt caaatgcatg ctcttcaacc tcaataacgt tatatcctga
     5701 ttcacgtaat atttttgggg taaatttagt tcctgttcca ttaactgcgc taaaaataat
     5761 ttttaaatct tttttagctt cttgctcttt tttgtacgtc tctgttttag agctagaaat
     5821 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtggt
     5881 gctttttttg ttttttatgt cttcgagtca tgtaattagt tatgtcacgc ttacgttcac
     5941 gccctccccc cacatccgct ctaaccgaaa aggaaggagt tagacaacct gaagtctagg
     6001 tccctattta tttttttata gttatgttag tattaagaac gttatttata tttcaaattt
     6061 ttcttttttt tctgtacaga cgcgtgtacg catgtaacat tatactgaaa accttgcttg
     6121 agaaggtttt gggacgctcg aaggctttaa tttgcggccg gtacccaatt cgccctatag
     6181 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     6241 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     6301 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggac
     6361 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     6421 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     6481 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     6541 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     6601 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     6661 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     6721 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     6781 gcgaatttta acaaaatatt aacgcttaca atttcctgat gcggtatttt ctccttacgc
     6841 atctgtgcgg tatttcacac cgcatagggt aataactgat ataattaaat tgaagctcta
     6901 atttgtgagt ttagtataca tgcatttact tataatacag ttttttagtt ttgctggccg
     6961 catcttctca aatatgcttc ccagcctgct tttctgtaac gttcaccctc taccttagca
     7021 tcccttccct ttgcaaatag tcctcttcca acaataataa tgtcagatcc tgtagagacc
     7081 acatcatcca cggttctata ctgttgaccc aatgcatctc ccttgtcatc taaacccaca
     7141 ccgggtgtca taatcaacca atcgtaacct tcatctcttc cacccatgtc tctttgagca
     7201 ataaagccga taacaaaatc tttgtcgctc ttcgcaatgt caacagtacc cttagtatat
     7261 tctccagtag atagggagcc cttgcatgac aattctgcta acatcaaaag gcctctaggt
     7321 tcctttgtta cttcttctgc cgcctgcttc aaaccgctaa caatacctgg gcccaccaca
     7381 ccgtgtgcat tcgtaatgtc tgcccattct gctattctgt atacacccgc agagtactgc
     7441 aatttgactg tattaccaat gtcagcaaat tttctgtctt cgaagagtaa aaaattgtac
     7501 ttggcggata atgcctttag cggcttaact gtgccctcca tggaaaaatc agtcaagata
     7561 tccacatgtg tttttagtaa acaaattttg ggacctaatg cttcaactaa ctccagtaat
     7621 tccttggtgg tacgaacatc caatgaagca cacaagtttg tttgcttttc gtgcatgata
     7681 ttaaatagct tggcagcaac aggactagga tgagtagcag cacgttcctt atatgtagct
     7741 ttcgacatga tttatcttcg tttcctgcag gtttttgttc tgtgcagttg ggttaagaat
     7801 actgggcaat ttcatgtttc ttcaacacta catatgcgta tatataccaa tctaagtctg
     7861 tgctccttcc ttcgttcttc cttctgttcg gagattaccg aatcaaaaaa atttcaagga
     7921 aaccgaaatc aaaaaaaaga ataaaaaaaa aatgatgaat tgaaaaggtg gtatggtgca
     7981 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     8041 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     8101 ccgtcgccgg gagctgcatg tgt
//
