LOCUS       sequence-325004-        6182 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CEN/ARS with P(Z)-mCherry controlled by the ZEM transcription 
            factor, also encode UBC9 sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6182)
  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 6182)
  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: pNTI763 p(Z)-mCherry + UBC9 sgRNA
FEATURES             Location/Qualifiers
     source          1..6182
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        358..462
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(463..531,532..1323)
                     /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(681..700)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1494..2082
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1983..2002
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(2233..2420)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2341..2365
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(2804..2823)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3073..3091
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(3260..3283)
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     protein_bind    4267..4285
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        4370..4445
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4458..4520
                     /label=RPR1 terminator
                     /note="transcription terminator for the S. cerevisiae RNase
                     P RNA gene"
     CDS             complement(4876..5679)
                     /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(5680..5895)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(5941..5959)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    join(6000..6182,1..321)
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
ORIGIN
        1 agcgtacttt acatatatat ttattagaca agaaaagcag attaaataga tatacattcg
       61 attaacgata agtaaaatgt aaaatcacag gattttcgtg tgtggtcttc tacacagaca
      121 agatgaaaca attcggcatt aatacctgag agcaggaaga gcaagataaa aggtagtatt
      181 tgttggcgat ccccctagag tcttttacat cttcggaaaa caaaaactat tttttcttta
      241 atttcttttt ttactttcta tttttaattt atatatttat attaaaaaat ttaaattata
      301 attattttta tagcacgtga tgaaaaggac ccaggtggca cttttcgggg aaatgtgcgc
      361 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
      421 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
      481 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
      541 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
      601 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
      661 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
      721 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
      781 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
      841 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
      901 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
      961 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     1021 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     1081 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     1141 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     1201 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     1261 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     1321 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     1381 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     1441 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     1501 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     1561 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     1621 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     1681 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     1741 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     1801 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     1861 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     1921 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     1981 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     2041 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     2101 tttttacggt tcctggcctt ttgctggcct tttgctggcc ttttgctggc cttttgctcg
     2161 ggggatctgc ctgagctcta atacgactca ctatagcaga tctatattac cctgttatcc
     2221 ctagcggatc tgccggtaga ggtgtggtca ataagagcga cctcatacta tacctgagaa
     2281 agcaacctga cctacaggaa agagttactc aagaataaga attttcgttt taaaacctaa
     2341 gagtcacttt aaaatttgta tacacttatt ttttttataa cttatttaat aataaaaatc
     2401 ataaatcata agaaattcgc ttatttagaa gtggcgcgca agcttacact ctttccctac
     2461 acgacgctct tccgatctgg gacactattc cgtgacgcgc ggggagatcc agtcactcgg
     2521 ccttgctatt tgtacaattc atccatacca ccagtagaat gtctaccttc agctctttca
     2581 tattgttcca caatggtgta gtcctcgttg tgggaggtga tgtccaactt gatgttgacg
     2641 ttgtaggcgc cgggcagctg cacgggcttc ttggccttgt aggtggtctt gacctcagcg
     2701 tcgtagtggc cgccgtcctt cagcttcagc ctctgcttga tctcgccctt cagggcgccg
     2761 tcctcggggt acattctctc ggaggaggcc tcccagcaca tggtcttctt ctgcattacg
     2821 gggccgtcgg aggggaagtt ggtgcctctc aacttcacct tgtagatgaa ctcgccgtcc
     2881 tgcagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt catcactctc
     2941 tcccacttga agccctcggg gaaggacagc ttcaagtagt cggggatgtc ggcggggtgc
     3001 ttcacgtagg ccttggagcc gtacatgaac tgaggggaca ggatgtccca ggcgaagggc
     3061 agggggccac ccttggtcac cttcagcttg gcggtctggg tgccctcgta gggtctgccc
     3121 tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat gtgcaccttg
     3181 aatctcatga actccttgat gatggccatg ttatcctcct cgcccttgct caccatgcta
     3241 gctatagttt tttctccttg acgttaaagt atagaggtat attaacaatt ttttgttgat
     3301 acttttatga catttgaata agaagtaata caaactgaaa atgttgaaag tattagttaa
     3361 agtggttatg cagcttttcc atttatatat ctgttaatag atcaaaaatc atcgcttcgc
     3421 tgattaatta ccccagaaat aaggctaaaa aactaatcgc attatcatcc tatggttgtt
     3481 aatttgattc gttaatttga aggtttgtgg ggccaggtta ctgccaattt ttcctcttca
     3541 taaccataaa agctagtatt gtagaatctt tattgttcgg agcagtgcgg cattaggcac
     3601 atctgcgttt caggaacgcg accggtgaag acgaggacgc acggtctaga cgcccacgca
     3661 cgcccacgcc cgcccacgca cgcccacgcc cgcccacgca cgcccacgcg gccgcacttc
     3721 aatatagcaa tgagcagtta agcgtattac tgaaagttcc aaagagaagg tttttttagg
     3781 ctaagataat ggggctcttt acatttccac aacatataag taagattaga tatggatatg
     3841 tatatggata tgtatatggt ggtaatgcca tgtaatatga ttattaaact tctttgcgtc
     3901 catccaaaaa aaaagtaaga atttttgaaa attcaatata acgatctgcc aattgaacat
     3961 aacatggtag ttacatatac tagtaatatg gttcggcaca cattaaaagt ataaaaacta
     4021 tctgaattac gaattacata tattggtcat aaaaatcaat caatcatcgt gtgttttata
     4081 tgtctcttat ctaagtataa gaatatccat agttaatatt cacttacgct accttttaac
     4141 ctgtaatcat tgtcaacagg atatgttaac gacccacatt gataaacgct agtatttctt
     4201 tttcctcttc ttattggccg gctgtctcta tactccccta tagtctgttt cttttcgttt
     4261 cgattgtccc tatcagtgat agagatggcg cacatggtac gctgtggtgc tcgcggctgg
     4321 gaacgaaact ctgggagctg cgattggcat aaatgtttgt tatggagacg ttttagagct
     4381 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
     4441 ggtgcttttt tctcgagcca tatccaactt ccaatttaat ctttcttttt taattttcac
     4501 ttatttgcga tacagaaaga aaaaagcgat agtaactatt gaattttgtt tggatttggt
     4561 tagattagat atggtttctc tttatattta catgctaaaa atgggctaca ccagagatac
     4621 ataattagat atatatacgc cagtacacct tatcggccca agccttgtcc caaggcagcg
     4681 ttttgttctt ggaaacgctg ccctacacgt tcgctatgcg gccggtattc agtttaaacc
     4741 cgctccctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcataggg
     4801 taataactga tataattaaa ttgaagctct aatttgtgag tttagtatac atgcatttac
     4861 ttataataca gttttttagt tttgctggcc gcatcttctc aaatatgctt cccagcctgc
     4921 ttttctgtaa cgttcaccct ctaccttagc atcccttccc tttgcaaata gtcctcttcc
     4981 aacaataata atgtcagatc ctgtagagac cacatcatcc acggttctat actgttgacc
     5041 caatgcgtct cccttgtcat ctaaacccac accgggtgtc ataatcaacc aatcgtaacc
     5101 ttcatctctt ccacccatgt ctctttgagc aataaagccg ataacaaaat ctttgtcgct
     5161 cttcgcaatg tcaacagtac ccttagtata ttctccagta gatagggagc ccttgcatga
     5221 caattctgct aacatcaaaa ggcctctagg ttcctttgtt acttcttctg ccgcctgctt
     5281 caaaccgcta acaatacctg ggcccaccac accgtgtgca ttcgtaatgt ctgcccattc
     5341 tgctattctg tatacacccg cagagtactg caatttgact gtattaccaa tgtcagcaaa
     5401 ttttctgtct tcgaagagta aaaaattgta cttggcggat aatgccttta gcggcttaac
     5461 tgtgccctcc atggaaaaat cagtcaagat atccacatgt gtttttagta aacaaatttt
     5521 gggacctaat gcttcaacta actccagtaa ttccttggtg gtacgaacat ccaatgaagc
     5581 acacaagttt gtttgctttt cgtgcatgat attaaatagc ttggcagcaa caggactagg
     5641 atgagtagca gcacgttcct tatatgtagc tttcgacatg atttatcttc gtttcctgca
     5701 ggtttttgtt ctgtgcagtt gggttaagaa tactgggcaa tttcatgttt cttcaacact
     5761 acatatgcgt atatatacca atctaagtct gtgctccttc cttcgttctt ccttctgttc
     5821 ggagattacc gaatcaaaaa aatttcaagg aaaccgaaat caaaaaaaag aataaaaaaa
     5881 aaatgatgaa ttgaagaatt cggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     5941 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgg
     6001 atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata atttgggaat
     6061 ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt aaataaagaa
     6121 ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa atttcaacaa
     6181 aa
//
