LOCUS       sequence-102313-        5230 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA with 1x PP7 for yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5230)
  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 5230)
  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: pJZC548
FEATURES             Location/Qualifiers
     source          1..5230
                     /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"
     terminator      508..527
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        complement(556..574)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(595..611)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(595..611)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(608..630)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    619..635
                     /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(643..649,650..667,668..673))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    688..709
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(826..843)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(997..1585)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(1756..2547,2548..2616))
                     /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     2379..2398
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2617..2721)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    2758..3261
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     3303..3321
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3359..3381)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3481..3500
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3525..3740
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             3741..4544
                     /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(4675..5130)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4812..4833)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5024..5043
                     /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 cggtgcaaca
      481 taaggagttt atatggaaac ccttatgttt ttttgttttt tatgtctctg cagagttcgg
      541 taccagcttt tgttcccttt agtgagggtt aatttcgagc ttggcgtaat catggtcata
      601 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
      661 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
      721 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
      781 acgcgcgggg agaggcggtt ttcgtattgg gcgctcttcc gcttcctcgc tcactgactc
      841 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
      901 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
      961 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcg gcccccctga
     1021 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     1081 ataccaggcg ttcccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     1141 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg
     1201 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     1261 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     1321 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     1381 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     1441 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     1501 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     1561 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     1621 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     1681 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     1741 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     1801 atttcgttca tccatagttg cctgactgcc cgtcgtgtag ataactacga tacgggaggg
     1861 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     1921 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     1981 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     2041 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     2101 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     2161 gttgtgaaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     2221 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     2281 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     2341 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     2401 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     2461 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     2521 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     2581 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     2641 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     2701 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgggt ccttttcatc
     2761 acgtgctata aaaataatta taatttaaat tttttaatat aaatatataa attaaaaata
     2821 gaaagtaaaa aaagaaatta aagaaaaaat agtttttgtt ttccgaagat gtaaaagact
     2881 ctagggggat cgccaacaaa tactaccttt tatcttgctc ttcctgctct caggtattaa
     2941 tgccgaattg tttcatcttg tctgtgtaga agaccacaca cgaaaatcct gtgattttac
     3001 attttactta tcgttaatcg aatgtatatc tatttaatct gcttttcttg tctaataaat
     3061 atatatgtaa agtacgcttt ttgttgaaat tttttaaacc tttgtttatt tttttttctt
     3121 cattccgtaa ctcttctacc ttctttattt actttctaaa atccaaatac aaaacataaa
     3181 aataaataaa cacagagtaa attcccaaat tattccatca ttaaaagata cgaggcgcgt
     3241 gtaagttaca ggcaagcgat ccgtcctaag aaaccattat tatcatgaca ttaacctata
     3301 aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc
     3361 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     3421 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg
     3481 cggcatcaga gcagattgta ctgagagtgc accacgcttt tcaattcaat tcatcatttt
     3541 ttttttattc ttttttttga tttcggtttc cttgaaattt ttttgattcg gtaatctccg
     3601 aacagaagga agaacgaagg aaggagcaca gacttagatt ggtatatata cgcatatgta
     3661 gtgttgaaga aacatgaaat tgcccagtat tcttaaccca actgcacaga acaaaaacgt
     3721 gcaggaaacg aagataaatc atgtcgaaag ctacatataa ggaacgtgct gctactcatc
     3781 ctagtcctgt tgctgccaag ctatttaata tcatgcacga aaagcaaaca aacttgtgtg
     3841 cttcattgga tgttcgtacc accaaggaat tactggagtt agttgaagca ttaggtccca
     3901 aaatttgttt actaaaaaca catgtggata tcttgactga tttttccatg gagggcacag
     3961 ttaagccgct aaaggcatta tccgccaagt acaatttttt actcttcgaa gacagaaaat
     4021 ttgctgacat tggtaataca gtcaaattgc agtactctgc gggtgtatac agaatagcag
     4081 aatgggcaga cattacgaat gcacacggtg tggtgggccc aggtattgtt agcggtttga
     4141 agcaggcggc agaagaagta acaaaggaac ctagaggcct tttgatgtta gcagaattgt
     4201 catgcaaggg ctccctatct actggagaat atactaaggg tactgttgac attgcgaaga
     4261 gcgacaaaga ttttgttatc ggctttattg ctcaaagaga catgggtgga agagatgaag
     4321 gttacgattg gttgattatg acacccggtg tgggtttaga tgacaaggga gacgcattgg
     4381 gtcaacagta tagaaccgtg gatgatgtgg tctctacagg atctgacatt attattgttg
     4441 gaagaggact atttgcaaag ggaagggatg ctaaggtaga gggtgaacgt tacagaaaag
     4501 caggctggga agcatatttg agaagatgcg gccagcaaaa ctaaaaaact gtattataag
     4561 taaatgcatg tatactaaac tcacaaatta gagcttcaat ttaattatat cagttattac
     4621 cctatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggaaattg
     4681 taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     4741 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     4801 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     4861 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     4921 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     4981 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     5041 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     5101 ccgcgcttaa tgcgccgcta cagggcgcgt cgcgccattc gccattcagg ctgcgcaact
     5161 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaggggggat
     5221 gtgctgcaag
//
