LOCUS       Exported                5278 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  sgRNA with 2x PP7 for yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    pJZC603
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5278)
  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 5278)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5278
                     /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"
     terminator      556..575
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        complement(604..622)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(643..659)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(643..659)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(656..678)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    667..683
                     /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(691..721)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    736..757
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(874..891)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1045..1633)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1804..2664)
                     /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     2427..2446
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2665..2769)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    2806..3309
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     3351..3369
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3407..3429)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3529..3548
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3573..3788
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             3789..4592
                     /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(4723..5178)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4860..4881)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5072..5091
                     /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 gctaaggagt ttatatggaa acccttagcc tgctgcgtaa ggagtttata tggaaaccct
      541 tacgcagcag ttcccttttt ttgtttttta tgtctctgca gagttcggta ccagcttttg
      601 ttccctttag tgagggttaa tttcgagctt ggcgtaatca tggtcatagc tgtttcctgt
      661 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
      721 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
      781 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
      841 aggcggtttt cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
      901 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
      961 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     1021 taaaaaggcc gcgttgctgg cgtttttcca taggctcggc ccccctgacg agcatcacaa
     1081 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     1141 cccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     1201 gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct
     1261 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     1321 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     1381 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     1441 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     1501 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     1561 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     1621 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     1681 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     1741 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     1801 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     1861 catagttgcc tgactgcccg tcgtgtagat aactacgata cgggagggct taccatctgg
     1921 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     1981 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     2041 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     2101 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     2161 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgaaaaaa
     2221 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     2281 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     2341 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     2401 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     2461 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     2521 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     2581 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     2641 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     2701 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     2761 ggttccgcgc acatttcccc gaaaagtgcc acctgggtcc ttttcatcac gtgctataaa
     2821 aataattata atttaaattt tttaatataa atatataaat taaaaataga aagtaaaaaa
     2881 agaaattaaa gaaaaaatag tttttgtttt ccgaagatgt aaaagactct agggggatcg
     2941 ccaacaaata ctacctttta tcttgctctt cctgctctca ggtattaatg ccgaattgtt
     3001 tcatcttgtc tgtgtagaag accacacacg aaaatcctgt gattttacat tttacttatc
     3061 gttaatcgaa tgtatatcta tttaatctgc ttttcttgtc taataaatat atatgtaaag
     3121 tacgcttttt gttgaaattt tttaaacctt tgtttatttt tttttcttca ttccgtaact
     3181 cttctacctt ctttatttac tttctaaaat ccaaatacaa aacataaaaa taaataaaca
     3241 cagagtaaat tcccaaatta ttccatcatt aaaagatacg aggcgcgtgt aagttacagg
     3301 caagcgatcc gtcctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     3361 tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     3421 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     3481 agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc
     3541 agattgtact gagagtgcac cacgcttttc aattcaattc atcatttttt ttttattctt
     3601 ttttttgatt tcggtttcct tgaaattttt ttgattcggt aatctccgaa cagaaggaag
     3661 aacgaaggaa ggagcacaga cttagattgg tatatatacg catatgtagt gttgaagaaa
     3721 catgaaattg cccagtattc ttaacccaac tgcacagaac aaaaacgtgc aggaaacgaa
     3781 gataaatcat gtcgaaagct acatataagg aacgtgctgc tactcatcct agtcctgttg
     3841 ctgccaagct atttaatatc atgcacgaaa agcaaacaaa cttgtgtgct tcattggatg
     3901 ttcgtaccac caaggaatta ctggagttag ttgaagcatt aggtcccaaa atttgtttac
     3961 taaaaacaca tgtggatatc ttgactgatt tttccatgga gggcacagtt aagccgctaa
     4021 aggcattatc cgccaagtac aattttttac tcttcgaaga cagaaaattt gctgacattg
     4081 gtaatacagt caaattgcag tactctgcgg gtgtatacag aatagcagaa tgggcagaca
     4141 ttacgaatgc acacggtgtg gtgggcccag gtattgttag cggtttgaag caggcggcag
     4201 aagaagtaac aaaggaacct agaggccttt tgatgttagc agaattgtca tgcaagggct
     4261 ccctatctac tggagaatat actaagggta ctgttgacat tgcgaagagc gacaaagatt
     4321 ttgttatcgg ctttattgct caaagagaca tgggtggaag agatgaaggt tacgattggt
     4381 tgattatgac acccggtgtg ggtttagatg acaagggaga cgcattgggt caacagtata
     4441 gaaccgtgga tgatgtggtc tctacaggat ctgacattat tattgttgga agaggactat
     4501 ttgcaaaggg aagggatgct aaggtagagg gtgaacgtta cagaaaagca ggctgggaag
     4561 catatttgag aagatgcggc cagcaaaact aaaaaactgt attataagta aatgcatgta
     4621 tactaaactc acaaattaga gcttcaattt aattatatca gttattaccc tatgcggtgt
     4681 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggaaattgta aacgttaata
     4741 ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg
     4801 aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc
     4861 cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa
     4921 ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt
     4981 cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac
     5041 ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta
     5101 gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg
     5161 cgccgctaca gggcgcgtcg cgccattcgc cattcaggct gcgcaactgt tgggaagggc
     5221 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa ggggggatgt gctgcaag
//
