LOCUS       sequence-324992-        7702 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For yeast genomic integration of sgRNA against RPC31.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7702)
  AUTHORS   Muller R, Meacham ZA, Ferguson L, Ingolia NT
  TITLE     CiBER-seq dissects genetic networks by quantitative CRISPRi 
            profiling of expression phenotypes.
  JOURNAL   Science. 2020 Dec 11;370(6522). pii: 370/6522/eabb9662. doi: 
            10.1126/science.abb9662.
  PUBMED    33303588
REFERENCE   2  (bases 1 to 7702)
  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: "Science."; 
            date: "2020-12-11"; volume: "370(6522). pii: 370/6522/eabb9662. 
            doi"; pages: " 10.1126/science.abb9662"
COMMENT     Sequence Label: pNTI733 pRPR1(TetO)-RPC31-sgRNA
FEATURES             Location/Qualifiers
     source          1..7702
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1862..1895
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1938..1957)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1939..1957)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1967..1983)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1976..1998)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2646..2679)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     primer_bind     complement(3149..3165)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3264..3281)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3435..4023)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3515..3534)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4145..4936,4937..5005))
                     /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     4768..4787
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5006..5097)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5139..5158
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5164..5183
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5390..5423
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     terminator      complement(6074..6239)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     6160..6184
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      6298..6487
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     protein_bind    6872..6890
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        6975..7050
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      7063..7125
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     protein_bind    7242..7275
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 agggaagcta gagatgcaga aggtaataaa cccaaagaac ctggaataac agaggcttca
       61 tcggagataa tatcaccaaa catgttgttg gtaataacaa caccgtttag cttagttggt
      121 gatttaacca aaatcatagc agcagagtcg atcaattggt gctgaacagt taattgtggg
      181 aactcagtct tgatggtttc ttcaacagtc tttctccaca atctggaaga ggcaagcacg
      241 ttagccttgt caagtgacca gattggtaat ggtgggtttt gttgcaatgc caagaaagca
      301 gccattcttg taattctttg aacttcagga acactgtatt tctcagagtc ccaagcaact
      361 ccgtcacctt catcttcttt tctttcacca aagtagatac caccaaccaa ttctctaacg
      421 acgacgaaat cggtaccctt tgcatattca ggcttcaaag gagaaagatc tagtaaagaa
      481 tcagaagcaa agttacatgg tcttaagttg gcgtatagac ccaattcctt tctgatcttc
      541 aatagacctt gttctggtct aactgcgccc gtaccccatt ttggaccacc aacagcacct
      601 agtaagacag catcggcttt cttagaggct tctagagctt catctggtaa aggagtgcca
      661 gtggcatcga tggcagcacc cccgatcaag tgatgttgga aattgaactt aatttctgga
      721 cggacttcag caatggcatt caagacctta atagcttcgt cagtaacttc tttaccgacg
      781 tgatcacccg gtaggacaac gatattctta gacatggtat aatctgtgta gtgtgggata
      841 ctttttactt cttcaaatag gtatcaacaa taaaaataaa tcgaagcaaa tgtaggaatg
      901 cgttaaagca gatgtactta cttagagtac atagatatat ttatataatt caatatataa
      961 aagtatatga agcatctgat gttgaacctg tcatgactct aaactggaat gggtagttat
     1021 gcttctgaag gttttccgtt agaaaatgag tgcagggatc aggcccggaa ccggttttag
     1081 cctgaaagga aaagaaaaaa atgcggtgat tactagcacg tgactgcgct gaattggaat
     1141 ccaccacaag tacagcgaat ggcaagaggg aaggggaata tattgtgcct ggcggtagtc
     1201 tgctatatca aatgttaaac ataactaaat gcagtagtga atagagtaat gtatttctac
     1261 atatgtgggt agggccaatg ggagcccgat gtctattgta cagatatttt ccttttatag
     1321 ttaatcaatt caacttggaa atcattgagt cgatatctac ttggataact ggagccgtgg
     1381 cagcgtttga aatttgacga gtctccttgt tttgttcgcg cattttcagt ttattcaaac
     1441 gggagatggc accgaatcca ctgacttctc tgttcaattt gaatggtaat aggaagaaag
     1501 tacatttcga gaaattttcg cccaacgtcg aaatctcatg gatgatatcc tcgatacaaa
     1561 tgacaccttc atcacctaat ttctcttcaa cgatattgtt gtcattcaag attatctctt
     1621 ttggttcttt atcttctggc ctttgccaca ttattctaga tctcttttgg attaaagaac
     1681 ggatagatgc aagtgaaggt tgtccgataa caatatatgg tgatacaagt ttcaacaatg
     1741 ggtacacgtc cttggtaagt ttgatgaaaa caccagtgtt caactcaaca agtctcaaaa
     1801 gagataacac tttgtaagcc tttgctggga tcttcacagc gagctctcga gaacccttaa
     1861 tataacttcg tataatgtat gctatacgaa gttattaggt gatatcagat ccactagtgg
     1921 cctatgcacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     1981 tacaacgtcg tgactgggaa aaccctggcg ttacccctgc aggactagtg ctgaggcatt
     2041 aattgtgtcc gcgtttctaa ggccactttt caatgaaacg gatattgata tgctagtaaa
     2101 aggacgagct caagagcgaa aatataagta aagaattcga gtgcacttgt ctccatgcag
     2161 caagatttca tatgagtctt ttttatcttt ttacttttta cattacacga tatgcacttt
     2221 atgaaaattt aacgaggttg gaagccggat aatcaaccaa aatcaggcac gaaggcacac
     2281 tcgtatatgc atgttgttga aactctgtta cgctgaacta acaatcacac atgtagaggt
     2341 caccgggaaa agttgcgacc ccatggaagg tcgatctctt cgtttggctt tgcttggctg
     2401 gcggcattgc gcttcttcgc ttatacccgt ctcttgacgc tcgagctcgt tcattgagat
     2461 acctttattc ttgcacattt tctggctttt ttcgctactc gggtacatgt aatcatgcac
     2521 acagaaggtg ctgtagggtg aaagttcctt tgtgctgtcg tttgttttta atgccaaact
     2581 ttccggtgat caataaccac ctcattaaga cctcagcgcg gccgcaaatt taaataaaat
     2641 gaagtgaagt tcctatactt tctagagaat aggaacttct atagtgagtc gaataagggc
     2701 gacacaaaat ttattctaaa tgcataataa atactgataa catcttatag tttgtattat
     2761 attttgtatt atcgttgaca tgtataattt tgatatcaaa aactgatttt ccctttatta
     2821 ttttcgagat ttattttctt aattctcttt aacaaactag aaatattgta tatacaaaaa
     2881 atcataaata atagatgaat agtttaatta taggtgttca tcaatcgaaa aagcaacgta
     2941 tcttatttaa agtgcgttgc ttttttctca tttataaggt taaataattc tcatatatca
     3001 agcaaagtga caggcgccct taaatattct gacaaatgct ctttccctaa actcccccca
     3061 taaaaaaacc cgccgaagcg ggtttttacg ttatttgcgg attaacgatt actcgttatc
     3121 agaaccgccc agggggcccg agcttaagac tggccgtcgt tttacaacac agaaagagtt
     3181 tgtagaaacg caaaaaggcc atccgtcagg ggccttctgc ttagtttgat gcctggcagt
     3241 tccctactct cgccttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3301 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3361 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     3421 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3481 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3541 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3601 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     3661 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     3721 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     3781 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     3841 ttgaagtggt gggctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     3901 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     3961 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4021 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga cgcgcgcgta
     4081 actcacgtta agggattttg gtcatgagct tgcgccgtcc cgtcaagtca gcgtaatgct
     4141 ctgcttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     4201 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     4261 gccccagcgc tgcgatgata ccgcgagaac cacgctcacc ggctccggat ttatcagcaa
     4321 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     4381 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     4441 gcaacgttgt tgccatcgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     4501 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     4561 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     4621 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     4681 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     4741 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4801 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4861 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4921 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4981 cgacacggaa atgttgaata ctcatattct tcctttttca atattattga agcatttatc
     5041 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     5101 gggtcagtgt tacaaccaat taaccaattc tgaacattat cgcgagccca tttatacctg
     5161 aatatggctc ataacacccc ttgtttgcct ggcggcagta gcgcggtggt cccacctgac
     5221 cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gactccccat
     5281 gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     5341 ctttcgcccg ggctaattat ggggtgtcgc ccttattcga ctctatagtg aagttcctat
     5401 tctctagaaa gtataggaac ttctgaagtg gggatttaaa tgcggccgcg ctgagggttt
     5461 aatcgagatc tttgtgttcg gttacccggc tcagatccta acttcttctt ttggtatgtt
     5521 tattcgtata agttactgtt gtccacaggc aatactctgc agaaaattaa aacggcatta
     5581 atgctaggac aaccagaatt gttactactg tatgtgcgat agttgataac tgcaacatta
     5641 tgcccggtat attctcaaaa aaccctatta ctgcatacga agaaatcgca agagaaatct
     5701 ttcggtttgg aaaagctcac tgtgaggttc cttggagcca atagtaatac agcacaatcc
     5761 aaggaaaaat ctggcctata tgcaaggaag gagagatagt caaaagcatt ctttccccta
     5821 gaagttggtg catatatggc atcgttaaaa catattaccc ccaaaatttc ttctctaaac
     5881 gatgtgcttg gcctttgttt tggtttttga tgtcggtcgt ttgaggcccc ttgcggaaaa
     5941 tcgagatcgc cgaatggcac gcgagggaag ggaaataagg tttaaaggca ctgaaacaat
     6001 aggcaagaag taggcgagag ccgacatacg agactattaa gtcctcagcg agctcgcatg
     6061 gaatgcgtgc gatgagcgac ctcatgctat acctgagaaa gcaacctgac ctacaggaaa
     6121 gagttactca agaataagaa ttttcgtttt aaaacctaag agtcacttta aaatttgtat
     6181 acacttattt tttttataac ttatttaata ataaaaatca taaatcataa gaaattcgct
     6241 tatttagaag tgtcaacaac gtatctacca acggaatgcg tgcgatcgcg tgcattcatc
     6301 cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt
     6361 tatagttatg ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta
     6421 cagacgcgtg tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg
     6481 ctcgaagatc gcgtcagctg aagaagacgt tatcggggcg cgcctgagat ccagtcactc
     6541 gggatccgat ctgccaattg aacataacat ggtagttaca tatactagta atatggttcg
     6601 gcacacatta aaagtataaa aactatctga attacgaatt acatatattg gtcataaaaa
     6661 tcaatcaatc atcgtgtgtt ttatatgtct cttatctaag tataagaata tccatagtta
     6721 atattcactt acgctacctt ttaacctgta atcattgtca acaggatatg ttaacgaccc
     6781 acattgataa acgctagtat ttctttttcc tcttcttatt ggccggctgt ctctatactc
     6841 ccctatagtc tgtttctttt cgtttcgatt gtccctatca gtgatagaga tggcgcacat
     6901 ggtacgctgt ggtgctcgcg gctgggaacg aaactctggg agctgcgatt ggcattctat
     6961 aggttgctgc gatggtttta gagctagaaa tagcaagtta aaataaggct agtccgttat
     7021 caacttgaaa aagtggcacc gagtcggtgc ttttttctcg agccatatcc aacttccaat
     7081 ttaatctttc ttttttaatt ttcacttatt tgcgatacag aaagaaaaaa gcgatagtaa
     7141 ctattgaatt ttgtttggat ttggttagat tagatatggt ttctctttat atttacatgc
     7201 taaaaatggg cttcgtacgc tgcaggtcga caacccttaa tataacttcg tataatgtat
     7261 gctatacgaa gttattaggt ctagagatcc gcaggctaac cggaacctgt attatttagt
     7321 ttatgctacg ttaaataaag acctttcgtt cacataactg aatgtgtaat ggccttgaga
     7381 tttcaagcat accaagttgg tggagacggg gtcgttacaa aagactcttt aagccaagat
     7441 ttccttgaca gccttggcga tagcatcgcc aacctcagtg gtagagttag aaccaccaag
     7501 gtcaccggtt ctgacacctg catccaagac atttctaaca gcttcttcaa gagccctacc
     7561 ttcttcaacc aaatccaagg ataacttcaa catcatagct gcagataaga tggtagcaat
     7621 tgggttaacc ttgtttgctg gtaaatctgg ggcagaacca tgacatggtt cgtacaaacc
     7681 gaatgccttg ttagtgtcag gt
//
