LOCUS       sequence-102231-        5255 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA with 2x MS2 (wt+f6) for yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5255)
  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 5255)
  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: pJZC588
FEATURES             Location/Qualifiers
     source          1..5255
                     /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      533..552
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        complement(581..599)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(620..636)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(620..636)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(633..655)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    644..660
                     /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(668..674,675..692,693..698))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    713..734
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(851..868)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1022..1610)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(1781..2572,2573..2641))
                     /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     2404..2423
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2642..2746)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    2783..3286
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     3328..3346
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3384..3406)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3506..3525
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3550..3765
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             3766..4569
                     /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(4700..5155)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4837..4858)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5049..5068
                     /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 tgcgactccc acagtcactg gggagtcttc cctttttttg
      541 ttttttatgt ctctgcagag ttcggtacca gcttttgttc cctttagtga gggttaattt
      601 cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
      661 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
      721 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
      781 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggttttcgt attgggcgct
      841 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
      901 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      961 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     1021 ttttccatag gctcggcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     1081 ggcgaaaccc gacaggacta taaagatacc aggcgttccc ccctggaagc tccctcgtgc
     1141 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     1201 gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     1261 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     1321 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     1381 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     1441 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     1501 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     1561 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     1621 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     1681 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     1741 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     1801 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctgcccgtcg
     1861 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1921 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1981 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     2041 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     2101 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     2161 caaggcgagt tacatgatcc cccatgttgt gaaaaaaagc ggttagctcc ttcggtcctc
     2221 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     2281 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     2341 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     2401 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     2461 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     2521 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     2581 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     2641 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     2701 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     2761 aagtgccacc tgggtccttt tcatcacgtg ctataaaaat aattataatt taaatttttt
     2821 aatataaata tataaattaa aaatagaaag taaaaaaaga aattaaagaa aaaatagttt
     2881 ttgttttccg aagatgtaaa agactctagg gggatcgcca acaaatacta ccttttatct
     2941 tgctcttcct gctctcaggt attaatgccg aattgtttca tcttgtctgt gtagaagacc
     3001 acacacgaaa atcctgtgat tttacatttt acttatcgtt aatcgaatgt atatctattt
     3061 aatctgcttt tcttgtctaa taaatatata tgtaaagtac gctttttgtt gaaatttttt
     3121 aaacctttgt ttattttttt ttcttcattc cgtaactctt ctaccttctt tatttacttt
     3181 ctaaaatcca aatacaaaac ataaaaataa ataaacacag agtaaattcc caaattattc
     3241 catcattaaa agatacgagg cgcgtgtaag ttacaggcaa gcgatccgtc ctaagaaacc
     3301 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
     3361 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     3421 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     3481 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccac
     3541 gcttttcaat tcaattcatc attttttttt tattcttttt tttgatttcg gtttccttga
     3601 aatttttttg attcggtaat ctccgaacag aaggaagaac gaaggaagga gcacagactt
     3661 agattggtat atatacgcat atgtagtgtt gaagaaacat gaaattgccc agtattctta
     3721 acccaactgc acagaacaaa aacgtgcagg aaacgaagat aaatcatgtc gaaagctaca
     3781 tataaggaac gtgctgctac tcatcctagt cctgttgctg ccaagctatt taatatcatg
     3841 cacgaaaagc aaacaaactt gtgtgcttca ttggatgttc gtaccaccaa ggaattactg
     3901 gagttagttg aagcattagg tcccaaaatt tgtttactaa aaacacatgt ggatatcttg
     3961 actgattttt ccatggaggg cacagttaag ccgctaaagg cattatccgc caagtacaat
     4021 tttttactct tcgaagacag aaaatttgct gacattggta atacagtcaa attgcagtac
     4081 tctgcgggtg tatacagaat agcagaatgg gcagacatta cgaatgcaca cggtgtggtg
     4141 ggcccaggta ttgttagcgg tttgaagcag gcggcagaag aagtaacaaa ggaacctaga
     4201 ggccttttga tgttagcaga attgtcatgc aagggctccc tatctactgg agaatatact
     4261 aagggtactg ttgacattgc gaagagcgac aaagattttg ttatcggctt tattgctcaa
     4321 agagacatgg gtggaagaga tgaaggttac gattggttga ttatgacacc cggtgtgggt
     4381 ttagatgaca agggagacgc attgggtcaa cagtatagaa ccgtggatga tgtggtctct
     4441 acaggatctg acattattat tgttggaaga ggactatttg caaagggaag ggatgctaag
     4501 gtagagggtg aacgttacag aaaagcaggc tgggaagcat atttgagaag atgcggccag
     4561 caaaactaaa aaactgtatt ataagtaaat gcatgtatac taaactcaca aattagagct
     4621 tcaatttaat tatatcagtt attaccctat gcggtgtgaa ataccgcaca gatgcgtaag
     4681 gagaaaatac cgcatcagga aattgtaaac gttaatattt tgttaaaatt cgcgttaaat
     4741 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     4801 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     4861 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     4921 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     4981 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     5041 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     5101 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcgcgc
     5161 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
     5221 ttacgccagc tggcgaaggg gggatgtgct gcaag
//
