LOCUS       sequence-324994-        5023 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CEN/ARS Parent vector for constructing barcoded-gRNA CiBER-seq 
            plasmid libraries.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5023)
  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 5023)
  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: pNTI743 dual-barcoded gRNA parent vector
FEATURES             Location/Qualifiers
     source          1..5023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(56..271)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(317..335)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    376..879
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        916..1020
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1021..1089,1090..1881)
                     /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(1239..1258)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2052..2640
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2541..2560
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2791..2978)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2899..2923
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    3680..3698
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        3769..3844
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      3857..3919
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     CDS             complement(join(4275..5023,1..55))
                     /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"
ORIGIN
        1 cagcaacagg actaggatga gtagcagcac gttccttata tgtagctttc gacatgattt
       61 atcttcgttt cctgcaggtt tttgttctgt gcagttgggt taagaatact gggcaatttc
      121 atgtttcttc aacactacat atgcgtatat ataccaatct aagtctgtgc tccttccttc
      181 gttcttcctt ctgttcggag attaccgaat caaaaaaatt tcaaggaaac cgaaatcaaa
      241 aaaaagaata aaaaaaaaat gatgaattga agaattcggt tttcaccgtc atcaccgaaa
      301 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
      361 atggtttctt agacggatcg cttgcctgta acttacacgc gcctcgtatc ttttaatgat
      421 ggaataattt gggaatttac tctgtgttta tttattttta tgttttgtat ttggatttta
      481 gaaagtaaat aaagaaggta gaagagttac ggaatgaaga aaaaaaaata aacaaaggtt
      541 taaaaaattt caacaaaaag cgtactttac atatatattt attagacaag aaaagcagat
      601 taaatagata tacattcgat taacgataag taaaatgtaa aatcacagga ttttcgtgtg
      661 tggtcttcta cacagacaag atgaaacaat tcggcattaa tacctgagag caggaagagc
      721 aagataaaag gtagtatttg ttggcgatcc ccctagagtc ttttacatct tcggaaaaca
      781 aaaactattt tttctttaat ttcttttttt actttctatt tttaatttat atatttatat
      841 taaaaaattt aaattataat tatttttata gcacgtgatg aaaaggaccc aggtggcact
      901 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
      961 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     1021 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     1081 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     1141 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     1201 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     1261 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     1321 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     1381 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     1441 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     1501 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     1561 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     1621 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     1681 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     1741 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     1801 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     1861 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     1921 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     1981 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     2041 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     2101 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     2161 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     2221 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     2281 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     2341 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     2401 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     2461 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     2521 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     2581 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     2641 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctggcctt
     2701 ttgctggcct tttgctcggg ggatctgcct gagctctaat acgactcact atagcagatc
     2761 tatattaccc tgttatccct agcggatctg ccggtagagg tgtggtcaat aagagcgacc
     2821 tcatactata cctgagaaag caacctgacc tacaggaaag agttactcaa gaataagaat
     2881 tttcgtttta aaacctaaga gtcactttaa aatttgtata cacttatttt ttttataact
     2941 tatttaataa taaaaatcat aaatcataag aaattcgctt atttagaagt ggcgcgcaag
     3001 cttccacatg tgcattgcct cggcgcgcca gagtgactgg atcgagctct ctgcttttgt
     3061 gcgcgtatgt ttatgtatgt acctctctct ctatttctat ttttaaacca ccctctcaat
     3121 aaaataaaaa taataaagta tttttaagga aaagacgtgt ttaagcactg actttatcta
     3181 ctttttgtac gttttcattg atataatgtg ttttgtctct cccttttcta cgaaaatttc
     3241 aaaaattgac caaaaaaagg aatatatata cgaaaaacta ttatatttat atatcatagt
     3301 gttgataaaa aatgtttatc cattggaccg tgtatgctat agtgagtcgt attacgatct
     3361 gccaattgaa cataacatgg tagttacata tactagtaat atggttcggc acacattaaa
     3421 agtataaaaa ctatctgaat tacgaattac atatattggt cataaaaatc aatcaatcat
     3481 cgtgtgtttt atatgtctct tatctaagta taagaatatc catagttaat attcacttac
     3541 gctacctttt aacctgtaat cattgtcaac aggatatgtt aacgacccac attgataaac
     3601 gctagtattt ctttttcctc ttcttattgg ccggctgtct ctatactccc ctatagtctg
     3661 tttcttttcg tttcgattgt ccctatcagt gatagagatg gcgcacatgg tacgctgtgg
     3721 tgctcgcggc tgggaacgaa actctgggag ctgcgattgg cagcctaggt tttagagcta
     3781 gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     3841 gtgctttttt ctcgagccat atccaacttc caatttaatc tttctttttt aattttcact
     3901 tatttgcgat acagaaagaa aaaagcgata gtaactattg aattttgttt ggatttggtt
     3961 agattagata tggtttctct ttatatttac atgctaaaaa tgggctacac cagagataca
     4021 taattagata tatatacgcc agtacacctt atcggcccaa gccttgtccc aaggcagcgt
     4081 tttgttcttg gaaacgctgc cctacacgtt cgctatgcgg ccggtattca gtttaaaccc
     4141 gctccctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatagggt
     4201 aataactgat ataattaaat tgaagctcta atttgtgagt ttagtataca tgcatttact
     4261 tataatacag ttttttagtt ttgctggccg catcttctca aatatgcttc ccagcctgct
     4321 tttctgtaac gttcaccctc taccttagca tcccttccct ttgcaaatag tcctcttcca
     4381 acaataataa tgtcagatcc tgtagagacc acatcatcca cggttctata ctgttgaccc
     4441 aatgcgtctc ccttgtcatc taaacccaca ccgggtgtca taatcaacca atcgtaacct
     4501 tcatctcttc cacccatgtc tctttgagca ataaagccga taacaaaatc tttgtcgctc
     4561 ttcgcaatgt caacagtacc cttagtatat tctccagtag atagggagcc cttgcatgac
     4621 aattctgcta acatcaaaag gcctctaggt tcctttgtta cttcttctgc cgcctgcttc
     4681 aaaccgctaa caatacctgg gcccaccaca ccgtgtgcat tcgtaatgtc tgcccattct
     4741 gctattctgt atacacccgc agagtactgc aatttgactg tattaccaat gtcagcaaat
     4801 tttctgtctt cgaagagtaa aaaattgtac ttggcggata atgcctttag cggcttaact
     4861 gtgccctcca tggaaaaatc agtcaagata tccacatgtg tttttagtaa acaaattttg
     4921 ggacctaatg cttcaactaa ctccagtaat tccttggtgg tacgaacatc caatgaagca
     4981 cacaagtttg tttgcttttc gtgcatgata ttaaatagct tgg
//
