LOCUS       sequence-111865-        5220 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA with 1x MS2 for yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5220)
  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 5220)
  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: pJZC545
FEATURES             Location/Qualifiers
     source          1..5220
                     /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        481..499
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     terminator      502..521
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        complement(550..568)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(589..605)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(589..605)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(602..624)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    613..629
                     /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(637..643,644..661,662..667))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    682..703
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(820..837)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(991..1579)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(1750..2541,2542..2610))
                     /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     2373..2392
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2611..2715)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    2752..3255
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     3297..3315
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3353..3375)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3475..3494
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3519..3734
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             3735..4538
                     /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(4669..5124)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4806..4827)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5018..5037
                     /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 cggtgcgcgc
      481 acatgaggat cacccatgtg ctttttttgt tttttatgtc tctgcagagt tcggtaccag
      541 cttttgttcc ctttagtgag ggttaatttc gagcttggcg taatcatggt catagctgtt
      601 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
      661 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
      721 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
      781 ggggagaggc ggttttcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
      841 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
      901 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
      961 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctcggccccc ctgacgagca
     1021 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     1081 ggcgttcccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     1141 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag
     1201 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     1261 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     1321 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     1381 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     1441 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     1501 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     1561 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     1621 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     1681 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     1741 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     1801 ttcatccata gttgcctgac tgcccgtcgt gtagataact acgatacggg agggcttacc
     1861 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     1921 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     1981 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     2041 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     2101 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     2161 aaaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     2221 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     2281 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     2341 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     2401 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     2461 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     2521 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     2581 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     2641 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     2701 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gggtcctttt catcacgtgc
     2761 tataaaaata attataattt aaatttttta atataaatat ataaattaaa aatagaaagt
     2821 aaaaaaagaa attaaagaaa aaatagtttt tgttttccga agatgtaaaa gactctaggg
     2881 ggatcgccaa caaatactac cttttatctt gctcttcctg ctctcaggta ttaatgccga
     2941 attgtttcat cttgtctgtg tagaagacca cacacgaaaa tcctgtgatt ttacatttta
     3001 cttatcgtta atcgaatgta tatctattta atctgctttt cttgtctaat aaatatatat
     3061 gtaaagtacg ctttttgttg aaatttttta aacctttgtt tatttttttt tcttcattcc
     3121 gtaactcttc taccttcttt atttactttc taaaatccaa atacaaaaca taaaaataaa
     3181 taaacacaga gtaaattccc aaattattcc atcattaaaa gatacgaggc gcgtgtaagt
     3241 tacaggcaag cgatccgtcc taagaaacca ttattatcat gacattaacc tataaaaata
     3301 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac
     3361 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     3421 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
     3481 cagagcagat tgtactgaga gtgcaccacg cttttcaatt caattcatca tttttttttt
     3541 attctttttt ttgatttcgg tttccttgaa atttttttga ttcggtaatc tccgaacaga
     3601 aggaagaacg aaggaaggag cacagactta gattggtata tatacgcata tgtagtgttg
     3661 aagaaacatg aaattgccca gtattcttaa cccaactgca cagaacaaaa acgtgcagga
     3721 aacgaagata aatcatgtcg aaagctacat ataaggaacg tgctgctact catcctagtc
     3781 ctgttgctgc caagctattt aatatcatgc acgaaaagca aacaaacttg tgtgcttcat
     3841 tggatgttcg taccaccaag gaattactgg agttagttga agcattaggt cccaaaattt
     3901 gtttactaaa aacacatgtg gatatcttga ctgatttttc catggagggc acagttaagc
     3961 cgctaaaggc attatccgcc aagtacaatt ttttactctt cgaagacaga aaatttgctg
     4021 acattggtaa tacagtcaaa ttgcagtact ctgcgggtgt atacagaata gcagaatggg
     4081 cagacattac gaatgcacac ggtgtggtgg gcccaggtat tgttagcggt ttgaagcagg
     4141 cggcagaaga agtaacaaag gaacctagag gccttttgat gttagcagaa ttgtcatgca
     4201 agggctccct atctactgga gaatatacta agggtactgt tgacattgcg aagagcgaca
     4261 aagattttgt tatcggcttt attgctcaaa gagacatggg tggaagagat gaaggttacg
     4321 attggttgat tatgacaccc ggtgtgggtt tagatgacaa gggagacgca ttgggtcaac
     4381 agtatagaac cgtggatgat gtggtctcta caggatctga cattattatt gttggaagag
     4441 gactatttgc aaagggaagg gatgctaagg tagagggtga acgttacaga aaagcaggct
     4501 gggaagcata tttgagaaga tgcggccagc aaaactaaaa aactgtatta taagtaaatg
     4561 catgtatact aaactcacaa attagagctt caatttaatt atatcagtta ttaccctatg
     4621 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggaa attgtaaacg
     4681 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
     4741 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
     4801 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
     4861 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
     4921 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
     4981 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
     5041 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
     5101 ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact gttgggaagg
     5161 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
//
