LOCUS       sequence-102234-        5268 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA with MS2-PP7 for yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5268)
  AUTHORS   Zalatan JG, Lee ME, Almeida R, Gilbert LA, Whitehead EH, La Russa M,
            Tsai JC, Weissman JS, Dueber JE, Qi LS, Lim WA
  TITLE     Engineering Complex Synthetic Transcriptional Programs with CRISPR 
            RNA Scaffolds.
  JOURNAL   Cell. 2014 Dec 18. pii: S0092-8674(14)01570-0. doi: 
            10.1016/j.cell.2014.11.052.
  PUBMED    25533786
REFERENCE   2  (bases 1 to 5268)
  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: "Cell. 2014 
            Dec 18. pii: S0092-8674(14)01570-0. doi: 
            10.1016/j.cell.2014.11.052."
COMMENT     Sequence Label: pJZC593
FEATURES             Location/Qualifiers
     source          1..5268
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..52
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     44..61
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     45..61
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     68..87
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        68..86
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        112..380
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        401..476
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        483..501
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     terminator      546..565
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        complement(594..612)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(633..649)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(633..649)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(646..668)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    657..673
                     /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)."
     promoter        complement(join(681..687,688..705,706..711))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    726..747
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(864..881)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1035..1623)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(1794..2585,2586..2654))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2417..2436
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2655..2759)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    2796..3299
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     3341..3359
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3397..3419)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3519..3538
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3563..3778
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             3779..4582
                     /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"
     rep_origin      complement(4713..5168)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4850..4871)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5062..5081
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag
       61 tgaattgtaa tacgactcac tatagggcga attggagctc actcggaatt ctctttgaaa
      121 agataatgta tgattatgct ttcactcata tttatacaga aacttgatgt tttctttcga
      181 gtatatacaa ggtgattaca tgtacgtttg aagtacaact ctagattttg tagtgccctc
      241 ttgggctagc ggtaaaggtg cgcatttttt cacaccctac aatgttctgt tcaaaagatt
      301 ttggtcaaac gctgtagaag tgaaagttgg tgcgcatgtt tcggcgttcg aaacttctcc
      361 gcagtgaaag ataaatgatc acttttctct atcactgata gttttagagc tagaaatagc
      421 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgcggga
      481 gcacatgagg atcacccatg tgccacgagt aaggagttta tatggaaacc cttactcgtg
      541 ttcccttttt ttgtttttta tgtctctgca gagttcggta ccagcttttg ttccctttag
      601 tgagggttaa tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
      661 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
      721 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
      781 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttt
      841 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
      901 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
      961 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     1021 gcgttgctgg cgtttttcca taggctcggc ccccctgacg agcatcacaa aaatcgacgc
     1081 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt cccccctgga
     1141 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     1201 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg
     1261 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     1321 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     1381 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     1441 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     1501 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     1561 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     1621 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     1681 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     1741 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     1801 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     1861 tgactgcccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     1921 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     1981 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     2041 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     2101 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     2161 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgaaaaaa agcggttagc
     2221 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     2281 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     2341 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     2401 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     2461 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     2521 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     2581 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     2641 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     2701 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     2761 acatttcccc gaaaagtgcc acctgggtcc ttttcatcac gtgctataaa aataattata
     2821 atttaaattt tttaatataa atatataaat taaaaataga aagtaaaaaa agaaattaaa
     2881 gaaaaaatag tttttgtttt ccgaagatgt aaaagactct agggggatcg ccaacaaata
     2941 ctacctttta tcttgctctt cctgctctca ggtattaatg ccgaattgtt tcatcttgtc
     3001 tgtgtagaag accacacacg aaaatcctgt gattttacat tttacttatc gttaatcgaa
     3061 tgtatatcta tttaatctgc ttttcttgtc taataaatat atatgtaaag tacgcttttt
     3121 gttgaaattt tttaaacctt tgtttatttt tttttcttca ttccgtaact cttctacctt
     3181 ctttatttac tttctaaaat ccaaatacaa aacataaaaa taaataaaca cagagtaaat
     3241 tcccaaatta ttccatcatt aaaagatacg aggcgcgtgt aagttacagg caagcgatcc
     3301 gtcctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     3361 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     3421 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     3481 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     3541 gagagtgcac cacgcttttc aattcaattc atcatttttt ttttattctt ttttttgatt
     3601 tcggtttcct tgaaattttt ttgattcggt aatctccgaa cagaaggaag aacgaaggaa
     3661 ggagcacaga cttagattgg tatatatacg catatgtagt gttgaagaaa catgaaattg
     3721 cccagtattc ttaacccaac tgcacagaac aaaaacgtgc aggaaacgaa gataaatcat
     3781 gtcgaaagct acatataagg aacgtgctgc tactcatcct agtcctgttg ctgccaagct
     3841 atttaatatc atgcacgaaa agcaaacaaa cttgtgtgct tcattggatg ttcgtaccac
     3901 caaggaatta ctggagttag ttgaagcatt aggtcccaaa atttgtttac taaaaacaca
     3961 tgtggatatc ttgactgatt tttccatgga gggcacagtt aagccgctaa aggcattatc
     4021 cgccaagtac aattttttac tcttcgaaga cagaaaattt gctgacattg gtaatacagt
     4081 caaattgcag tactctgcgg gtgtatacag aatagcagaa tgggcagaca ttacgaatgc
     4141 acacggtgtg gtgggcccag gtattgttag cggtttgaag caggcggcag aagaagtaac
     4201 aaaggaacct agaggccttt tgatgttagc agaattgtca tgcaagggct ccctatctac
     4261 tggagaatat actaagggta ctgttgacat tgcgaagagc gacaaagatt ttgttatcgg
     4321 ctttattgct caaagagaca tgggtggaag agatgaaggt tacgattggt tgattatgac
     4381 acccggtgtg ggtttagatg acaagggaga cgcattgggt caacagtata gaaccgtgga
     4441 tgatgtggtc tctacaggat ctgacattat tattgttgga agaggactat ttgcaaaggg
     4501 aagggatgct aaggtagagg gtgaacgtta cagaaaagca ggctgggaag catatttgag
     4561 aagatgcggc cagcaaaact aaaaaactgt attataagta aatgcatgta tactaaactc
     4621 acaaattaga gcttcaattt aattatatca gttattaccc tatgcggtgt gaaataccgc
     4681 acagatgcgt aaggagaaaa taccgcatca ggaaattgta aacgttaata ttttgttaaa
     4741 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     4801 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     4861 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     4921 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     4981 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     5041 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     5101 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     5161 gggcgcgtcg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     5221 ggcctcttcg ctattacgcc agctggcgaa ggggggatgt gctgcaag
//
