LOCUS       sequence-112075-        8908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA with 1x MS2, Pol II promoter with ribozyme-gRNA-ribozyme 
            design for yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8908)
  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 8908)
  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: pJZC625
FEATURES             Location/Qualifiers
     source          1..8908
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        245..461
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     promoter        1625..2329
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     ncRNA           2360..2396
                     /product="self-cleaving hammerhead ribozyme (Ferre-D'Amare 
                     and Scott, 2010)"
                     /label=hammerhead ribozyme
                     /ncRNA_class="hammerhead_ribozyme"
     misc_RNA        2417..2492
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_RNA        2497..2515
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     ncRNA           2522..2589
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     promoter        3128..3471
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      4290..4487
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(4916..4935)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4917..4935)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4942..4959)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4942..4958)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4951..4973)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5099..5554
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5186..5205)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5396..5417
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5910..5929)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6029..6051
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6089..6107)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    6171..6674
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        6731..6835
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6836..6904,6905..7696)
                     /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(7054..7073)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7867..8455
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8356..8375
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8609..8626
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8743..8764
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8779..8784,8785..8802,8803..8809)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8817..8833
                     /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)."
     primer_bind     8822..8844
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8841..8857
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8841..8857
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        8878..8896
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 ggccggccat ttaaatgttt aaacctactg cgccaattga tgacaataca gacgatgata
       61 acaaaccgaa gttatctgat gtagaaaagg attaaagatg ctaagagata gtgatgatat
      121 ttcataaata atgtaattct atatatgtta attacctttt ttgcgaggca tatttatggt
      181 gaaggataag ttttgaccat caaagaaggt taatgtggct gtggtttcag ggtccataaa
      241 gcttttcaat tcatcttttt tttttttgtt cttttttttg attccggttt ctttgaaatt
      301 tttttgattc ggtaatctcc gagcagaagg aagaacgaag gaaggagcac agacttagat
      361 tggtatatat acgcatatgt ggtgttgaag aaacatgaaa ttgcccagta ttcttaaccc
      421 aactgcacag aacaaaaacc tgcaggaaac gaagataaat catgtcgaaa gctacatata
      481 aggaacgtgc tgctactcat cctagtcctg ttgctgccaa gctatttaat atcatgcacg
      541 aaaagcaaac aaacttgtgt gcttcattgg atgttcgtac caccaaggaa ttactggagt
      601 tagttgaagc attaggtccc aaaatttgtt tactaaaaac acatgtggat atcttgactg
      661 atttttccat ggagggcaca gttaagccgc taaaggcatt atccgccaag tacaattttt
      721 tactcttcga agacagaaaa tttgctgaca ttggtaatac agtcaaattg cagtactctg
      781 cgggtgtata cagaatagca gaatgggcag acattacgaa tgcacacggt gtggtgggcc
      841 ctaaaacaag aagagggttg actacatcac gatgaggggg atcgaagaaa tgatggtaaa
      901 tgaaatagga aatcaaggag catgaaggca aaagacaaat ataagggtcg aacgaaaaat
      961 aaagtgaaaa gtgttgatat gatgtatttg gctttgcggc gccgaaaaaa cgagtttacg
     1021 caattgcaca atcatgctga ctctgtggcg gacccgcgct cttgccggcc cggcgataac
     1081 gctgggcgtg aggctgtgcc cggcggagtt ttttgcgcct gcattttcca aggtttaccc
     1141 tgcgctaagg ggcgagattg gagaagcaat aagaatgccg gttggggttg cgatgatgac
     1201 gaccacgaca actggtgtca ttatttaagt tgccgaaaga acctgagtgc atttgcaaca
     1261 tgagtatact agaagaatga gccaagactt gcgagacgcg agtttgccgg tggtgcgaac
     1321 aatagagcga ccatgacctt gaaggtgaga cgcgcataac cgctagagta ctttgaagag
     1381 gaaacagcaa tagggttgct accagtataa atagacaggt acatacaaca ctggaaatgg
     1441 ttgtctgttt gagtacgctt tcaattcatt tgggtgtgca ctttattatg ttacaatatg
     1501 gaagggaact ttacacttct cctatgcaca tatattaatt aaagtccaat gctagtagag
     1561 aaggggggta acacccctcc gcgctctttt ccgatttttt tctaaaccgt ggaatatttc
     1621 ggatatcctt ttgttgtttc cgggtgtaca atatggactt cctcttttct ggcaaccaaa
     1681 cccatacatc gggattccta taataccttc gttggtctcc ctaacatgta ggtggcggag
     1741 gggagatata caatagaaca gataccagac aggacataat gggctaaaca agactacacc
     1801 aattacactg cctcattgat ggtggtacat aacgaactaa tactgtagcc ctagacttga
     1861 tagccatcat catatcgaag tttcactacc ctttttccat ttgccatcta ttgaagtaat
     1921 aataggcgca tgcaacttct tttctttttt tttcttttct ctctcccccg ttgttgtctc
     1981 accatatccg caatgacaaa aaaatgatgg aagacactaa aggaaaaaat taacgacaaa
     2041 gacagcacca acagatgtcg ttgttccaga gctgatgagg ggtatctcga agcacacgaa
     2101 actttttcct tccttcattc acgcacacta ctctctaatg agcaacggta tacggccttc
     2161 cttccagtta cttgaatttg aaataaaaaa aagtttgctg tcttgctatc aagtataaat
     2221 agacctgcaa ttattaatct tttgtttcct cgtcattgtt ctcgttccct ttcttccttg
     2281 tttctttttc tgcacaatat ttcaagctat accaagcata caatcaacta tctcatatac
     2341 aatgtctctc gagaaaagtc tgatgagtcc gtgaggacga aacgagtaag ctcgtcactt
     2401 ttctctatca ctgatagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     2461 atcaacttga aaaagtggca ccgagtcggt gcgcgcacat gaggatcacc catgtgcttt
     2521 tggccggcat ggtcccagcc tcctcgctgg cgccggctgg gcaacatgct tcggcatggc
     2581 gaatgggacg cggccgccac cgcggtggag ctctaagcaa atagctaaat tatatacgaa
     2641 ttaatattat gattaagtgt ttacgtgagt gcgatatttt tattactatc ttatacagtt
     2701 gtatatactc tataaaatga gttgtctatt aattaacgcg atgaatgctt tctgggttta
     2761 cctctccaac aactctagtt tacttctcaa tacattcaat tgtatttgat ttgtcaatac
     2821 ttcatcatta atcaattcta tagttttgtt tttctcgttt atttccaaat ttaatgcatc
     2881 aattttatta ttcaatttgt cgttgatttt ggttaatgat tttatggttt gatctctggc
     2941 attgattgtt tgtgttagtt tttcattatt gataattaaa ttatttaagt tagttatcaa
     3001 ctcggtgttt tcaagtttca agttttcaat ttctttagag tttattagat ttgtcaaagt
     3061 ttctgaattg cttgattggt ccagatctgt ttagcttgcc tcgtccccgc cgggtcaccc
     3121 ggccagcgac atggaggccc agaataccct ccttgacagt cttgacgtgc gcagctcagg
     3181 ggcatgatgt gactgtcgcc cgtacattta gcccatacat ccccatgtat aatcatttgc
     3241 atccatacat tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctgcagac
     3301 ctgcgagcag ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct
     3361 gtagagaaat ataaaaggtt aggatttgcc actgaggttc ttctttcata tacttccttt
     3421 taaaatcttg ctaggataca gttctcacat cacatccgaa cataaacaac catgacagtc
     3481 aacactaaga cctatagtga gagagcagaa actcatgcct caccagtagc acagcgatta
     3541 tttcgattaa tggaactgaa gaaaaccaat ttatgtgcat caattgacgt tgataccact
     3601 aaggaattcc ttgaattaat tgataaatta ggtccttatg tatgcttaat caagactcat
     3661 attgatataa tcaatgattt ttcctatgaa tccactattg aaccattatt agaactttca
     3721 cgtaaacatc aatttatgat ttttgaagat agaaaatttg ctgatattgg taataccgta
     3781 aagaaacaat atattggtgg agtttataaa attagtagtt gggcagatat taccaatgct
     3841 catggtgtca ctgggaatgg agtggttgaa ggattaaaac agggagctaa agaaaccacc
     3901 accaaccaag agccaagagg gttattgatg ttagctgaat tatcatcagt gggatcatta
     3961 gcatatggag aatattctca aaaaactgtt gaaattgcta aatccgataa ggaatttgtt
     4021 attggattta ttgcccaacg tgatatgggt ggccaagaag aaggatttga ttggcttatt
     4081 atgacacctg gagttggatt agatgataaa ggtgatggat taggacaaca atatagaact
     4141 gttgatgaag ttgttagcac tggaactgat attatcattg ttggtagagg attgtttggt
     4201 aaaggaagag atccagatat tgaaggtaaa aggtatagaa atgctggttg gaatgcttat
     4261 ttgaaaaaga ctggccaatt ataatcagta ctgacaataa aaagattctt gttttcaaga
     4321 acttgtcatt tgtatagttt ttttatattg tagttgttct attttaatca aatgttagcg
     4381 tgatttatat tttttttcgc ctcgacatca tctgcccaga tgcgaagtta agtgcgcaga
     4441 aagtaatatc atgcgtcaat cgtatgtgaa tgctggtcgc tatactgctg tcgattcgat
     4501 actaacgccg ccatccagtg tcgaaaacga gctcgaattc atcgacccgg gaatctcggt
     4561 cgtaatgatt tctataatga cgaaaaaaaa aaaattggaa agaaaaagct tcatggcctt
     4621 tataaaaagg aactatccaa tacctcgcca gaaccaagta acagtatttt acggggcaca
     4681 aatcaagaac aataagacag gactgtaaag atggacgcat tgaactccaa agaacaacaa
     4741 gagttccaaa aagtagtgga acaaaagcaa atgaaggatt tcatgcgttt gtactctaat
     4801 ctggtagaaa gatgtttcac agactgtgtc aatgacttca caacatcaaa gctaaccaat
     4861 aaggaacaaa catgcatcat gaagtgcgtt taaacggcca cgctggccca attcgcccta
     4921 tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     4981 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     5041 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggac
     5101 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     5161 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     5221 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     5281 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     5341 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     5401 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     5461 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     5521 gcgaatttta acaaaatatt aacgcttaca atttcctgat gcggtatttt ctccttacgc
     5581 atctgtgcgg tatttcacac cgcatagggt aataactgat ataattaaat tgaagctcta
     5641 atttgtgagt ttagtattag gatctctacc cttggcgaaa agtcctctgc caacaatgat
     5701 gatatctgat ccaccactta caacttcgtc gacggttctg tactgctgac ccaatatgcg
     5761 tatatatacc aatctaagtc tgtgctcctt ccttcgttct tccttctgtt cggagattac
     5821 cgaatcaaaa aaatttcaag gaaaccgaaa tcaaaaaaaa gaataaaaaa aaaatgatga
     5881 attgaaaagg tggtatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5941 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     6001 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     6061 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     6121 gtcatgataa taatggtttc ttagacgtcg gcgcgccctg ggtccttttc atcacgtgct
     6181 ataaaaataa ttataattta aattttttaa tataaatata taaattaaaa atagaaagta
     6241 aaaaaagaaa ttaaagaaaa aatagttttt gttttccgaa gatgtaaaag actctagggg
     6301 gatcgccaac aaatactacc ttttatcttg ctcttcctgc tctcaggtat taatgccgaa
     6361 ttgtttcatc ttgtctgtgt agaagaccac acacgaaaat cctgtgattt tacattttac
     6421 ttatcgttaa tcgaatgtat atctatttaa tctgcttttc ttgtctaata aatatatatg
     6481 taaagtacgc tttttgttga aattttttaa acctttgttt attttttttt cttcattccg
     6541 taactcttct accttcttta tttactttct aaaatccaaa tacaaaacat aaaaataaat
     6601 aaacacagag taaattccca aattattcca tcattaaaag atacgaggcg cgtgtaagtt
     6661 acaggcaagc gatccgtcct aagaaaccag gcgcgccagg acgtcaggtg gcacttttcg
     6721 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     6781 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     6841 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     6901 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     6961 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     7021 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     7081 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     7141 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     7201 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     7261 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     7321 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     7381 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     7441 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     7501 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     7561 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     7621 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     7681 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     7741 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     7801 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     7861 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     7921 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     7981 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     8041 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     8101 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     8161 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     8221 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     8281 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     8341 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     8401 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     8461 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     8521 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     8581 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     8641 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     8701 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     8761 tcattaggca ccccaggctt tacactttat gcttccggct cctatgttgt gtggaattgt
     8821 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agctcggaat
     8881 taaccctcac taaagggaac aaaagctg
//
