LOCUS       Exported                5266 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  sgRNA with 2x MS2 for yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    pJZC583
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5266)
  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 5266)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5266
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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"
     misc_RNA        512..530
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     terminator      544..563
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        complement(592..610)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(631..647)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(631..647)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(644..666)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    655..671
                     /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(679..709)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    724..745
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(862..879)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1033..1621)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1792..2652)
                     /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     2415..2434
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2653..2757)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    2794..3297
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     3339..3357
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3395..3417)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3517..3536
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3561..3776
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             3777..4580
                     /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(4711..5166)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4848..4869)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5060..5079
                     /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 tgccacgagc gacatgagga tcacccatgt cgctcgtgtt
      541 cccttttttt gttttttatg tctctgcaga gttcggtacc agcttttgtt ccctttagtg
      601 agggttaatt tcgagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
      661 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
      721 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
      781 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggttttcg
      841 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
      901 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
      961 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     1021 gttgctggcg tttttccata ggctcggccc ccctgacgag catcacaaaa atcgacgctc
     1081 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgttcc cccctggaag
     1141 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     1201 cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta
     1261 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     1321 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     1381 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     1441 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     1501 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     1561 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     1621 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     1681 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     1741 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     1801 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     1861 actgcccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     1921 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     1981 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     2041 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     2101 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     2161 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgaaaaaaag cggttagctc
     2221 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     2281 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     2341 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     2401 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     2461 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     2521 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     2581 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     2641 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     2701 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     2761 atttccccga aaagtgccac ctgggtcctt ttcatcacgt gctataaaaa taattataat
     2821 ttaaattttt taatataaat atataaatta aaaatagaaa gtaaaaaaag aaattaaaga
     2881 aaaaatagtt tttgttttcc gaagatgtaa aagactctag ggggatcgcc aacaaatact
     2941 accttttatc ttgctcttcc tgctctcagg tattaatgcc gaattgtttc atcttgtctg
     3001 tgtagaagac cacacacgaa aatcctgtga ttttacattt tacttatcgt taatcgaatg
     3061 tatatctatt taatctgctt ttcttgtcta ataaatatat atgtaaagta cgctttttgt
     3121 tgaaattttt taaacctttg tttatttttt tttcttcatt ccgtaactct tctaccttct
     3181 ttatttactt tctaaaatcc aaatacaaaa cataaaaata aataaacaca gagtaaattc
     3241 ccaaattatt ccatcattaa aagatacgag gcgcgtgtaa gttacaggca agcgatccgt
     3301 cctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     3361 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     3421 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     3481 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
     3541 gagtgcacca cgcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc
     3601 ggtttccttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg
     3661 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc
     3721 cagtattctt aacccaactg cacagaacaa aaacgtgcag gaaacgaaga taaatcatgt
     3781 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat
     3841 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca
     3901 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg
     3961 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg
     4021 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca
     4081 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac
     4141 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa
     4201 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg
     4261 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct
     4321 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac
     4381 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg
     4441 atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa
     4501 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa
     4561 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac
     4621 aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac
     4681 agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat
     4741 tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa
     4801 tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca
     4861 agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg
     4921 gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta
     4981 aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg
     5041 cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa
     5101 gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg
     5161 gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     5221 cctcttcgct attacgccag ctggcgaagg ggggatgtgc tgcaag
//
