LOCUS       sequence-264563-        5300 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Helper plasmid for SpCas9 gRNA cloning and expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5300)
  AUTHORS   Lian J, Schultz C, Cao M, HamediRad M, Zhao H
  TITLE     Multi-functional genome-wide CRISPR system for high throughput 
            genotype-phenotype mapping.
  JOURNAL   Nat Commun. 2019 Dec 19;10(1):5794. doi: 10.1038/s41467-019-13621-4.
  PUBMED    31857575
REFERENCE   2  (bases 1 to 5300)
  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: "Nat 
            Commun."; date: "2019-12-19"; volume: "10(1):5794. doi"; pages: " 
            10.1038/s41467-019-13621-4"
COMMENT     Sequence Label: p426*-SpSgH
FEATURES             Location/Qualifiers
     source          1..5300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        196..416
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             417..1220
                     /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"
     promoter        1309..1577
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        1594..1669
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1681..1700
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(1738..1754)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1738..1754)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1751..1773)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1762..1778
                     /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(1786..1792,1793..1810,1811..1816))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1831..1852
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1969..1986)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2140..2728)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2220..2239)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2899..3690,3691..3759))
                     /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     3522..3541
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3760..3864)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3891..5233
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(4854..4901)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     5273..5291
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc
      241 ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg
      301 agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc
      361 cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt
      421 cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat
      481 ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca
      541 aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg
      601 tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg
      661 ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca
      721 aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac
      781 acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa
      841 aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg
      901 gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct
      961 ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac
     1021 ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg
     1081 atgtcgtttc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa
     1141 gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa
     1201 gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac
     1261 aaattagagc ttcaatttaa ttatatcagt tattaccctg tgctcgagtc tttgaaaaga
     1321 taatgtatga ttatgctttc actcatattt atacagaaac ttgatgtttt ctttcgagta
     1381 tatacaaggt gattacatgt acgtttgaag tacaactcta gattttgtag tgccctcttg
     1441 ggctagcggt aaaggtgcgc attttttcac accctacaat gttctgttca aaagattttg
     1501 gtcaaacgct gtagaagtga aagttggtgc gcatgtttcg gcgttcgaaa cttctccgca
     1561 gtgaaagata aatgatcgga gaccgaggtc tccgttttag agctagaaat agcaagttaa
     1621 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgatcc
     1681 tttttttgtt ttttatgtct gagctccctg caggcatgca agcttggcgt aatcatggtc
     1741 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     1801 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     1861 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     1921 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     1981 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     2041 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     2101 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     2161 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     2221 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     2281 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     2341 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     2401 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2461 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     2521 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     2581 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     2641 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     2701 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     2761 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     2821 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     2881 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     2941 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3001 gggcttacca tctggcccca gtgctgcaat gataccgcgg ctcccacgct caccggctcc
     3061 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     3121 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     3181 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     3241 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     3301 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     3361 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     3421 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     3481 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     3541 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     3601 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     3661 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     3721 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     3781 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     3841 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg aacgaagcat
     3901 ctgtgcttca ttttgtagaa caaaaatgca acgcgagagc gctaattttt caaacaaaga
     3961 atctgagctg catttttaca gaacagaaat gcaacgcgaa agcgctattt taccaacgaa
     4021 gaatctgtgc ttcatttttg taaaacaaaa atgcaacgcg agagcgctaa tttttcaaac
     4081 aaagaatctg agctgcattt ttacagaaca gaaatgcaac gcgagagcgc tattttacca
     4141 acaaagaatc tatacttctt ttttgttcta caaaaatgca tcccgagagc gctatttttc
     4201 taacaaagca tcttagatta ctttttttct cctttgtgcg ctctataatg cagtctcttg
     4261 ataacttttt gcactgtagg tccgttaagg ttagaagaag gctactttgg tgtctatttt
     4321 ctcttccata aaaaaagcct gactccactt cccgcgttta ctgattacta gcgaagctgc
     4381 gggtgcattt tttcaagata aaggcatccc cgattatatt ctataccgat gtggattgcg
     4441 catactttgt gaacagaaag tgatagcgtt gatgattctt cattggtcag aaaattatga
     4501 acggtttctt ctattttgtc tctatatact acgtatagga aatgtttaca ttttcgtatt
     4561 gttttcgatt cactctatga atagttctta ctacaatttt tttgtctaaa gagtaatact
     4621 agagataaac ataaaaaatg tagaggtcga gtttagatgc aagttcaagg agcgaaaggt
     4681 ggatgggtag gttatatagg gatatagcac agagatatat agcaaagaga tacttttgag
     4741 caatgtttgt ggaagcggta ttcgcaatat tttagtagct cgttacagtc cggtgcgttt
     4801 ttggtttttt gaaagtgcgt cttcagagcg cttttggttt tcaaaagcgc tctgaagttc
     4861 ctatactttc tagagaatag gaacttcgga ataggaactt caaagcgttt ccgaaaacga
     4921 gcgcttccga aaatgcaacg cgagctgcgc acatacagct cactgttcac gtcgcaccta
     4981 tatctgcgtg ttgcctgtat atatatatac atgagaagaa cggcatagtg cgtgtttatg
     5041 cttaaatgcg tacttatatg cgtctattta tgtaggatga aaggtagtct agtacctcct
     5101 gtgatattat cccattccat gcggggtatc gtatgcttcc ttcagcacta ccctttagct
     5161 gttctatatg ctgccactcc tcaattggat tagtctcatc cttcaatgct atcatttcct
     5221 ttgatattgg atcatctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     5281 tatcacgagg ccctttcgtc
//
