LOCUS       sequence-269839-        5294 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 5294)
  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 5294)
  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..5294
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..66)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     166..185
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        209..424
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             425..1228
                     /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        1317..1585
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        1602..1677
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1689..1708
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(1746..1762)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1746..1762)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1759..1781)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1770..1786
                     /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(1794..1800,1801..1818,1819..1824))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1839..1860
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1977..1994)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2148..2736)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2228..2247)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2907..3698,3699..3767))
                     /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     3530..3549
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3768..3872)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3899..5241
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(4862..4909)
                     /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     join(5282..5294,1..6)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
       61 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
      121 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
      181 ttgtactgag agtgcaccat accacctttt caattcatca tttttttttt attctttttt
      241 ttgatttcgg tttccttgaa atttttttga ttcggtaatc tccgaacaga aggaagaacg
      301 aaggaaggag cacagactta gattggtata tatacgcata tgtagtgttg aagaaacatg
      361 aaattgccca gtattcttaa cccaactgca cagaacaaaa acctgcagga aacgaagata
      421 aatcatgtcg aaagctacat ataaggaacg tgctgctact catcctagtc ctgttgctgc
      481 caagctattt aatatcatgc acgaaaagca aacaaacttg tgtgcttcat tggatgttcg
      541 taccaccaag gaattactgg agttagttga agcattaggt cccaaaattt gtttactaaa
      601 aacacatgtg gatatcttga ctgatttttc catggagggc acagttaagc cgctaaaggc
      661 attatccgcc aagtacaatt ttttactctt cgaagacaga aaatttgctg acattggtaa
      721 tacagtcaaa ttgcagtact ctgcgggtgt atacagaata gcagaatggg cagacattac
      781 gaatgcacac ggtgtggtgg gcccaggtat tgttagcggt ttgaagcagg cggcagaaga
      841 agtaacaaag gaacctagag gccttttgat gttagcagaa ttgtcatgca agggctccct
      901 atctactgga gaatatacta agggtactgt tgacattgcg aagagcgaca aagattttgt
      961 tatcggcttt attgctcaaa gagacatggg tggaagagat gaaggttacg attggttgat
     1021 tatgacaccc ggtgtgggtt tagatgacaa gggagacgca ttgggtcaac agtatagaac
     1081 cgtggatgat gtcgtttcta caggatctga cattattatt gttggaagag gactatttgc
     1141 aaagggaagg gatgctaagg tagagggtga acgttacaga aaagcaggct gggaagcata
     1201 tttgagaaga tgcggccagc aaaactaaaa aactgtatta taagtaaatg catgtatact
     1261 aaactcacaa attagagctt caatttaatt atatcagtta ttaccctgtg ctcgagtctt
     1321 tgaaaagata atgtatgatt atgctttcac tcatatttat acagaaactt gatgttttct
     1381 ttcgagtata tacaaggtga ttacatgtac gtttgaagta caactctaga ttttgtagtg
     1441 ccctcttggg ctagcggtaa aggtgcgcat tttttcacac cctacaatgt tctgttcaaa
     1501 agattttggt caaacgctgt agaagtgaaa gttggtgcgc atgtttcggc gttcgaaact
     1561 gctccgcagt gaaagataaa tgatcggaga ccgaggtctc cgttttagag ctagaaatag
     1621 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     1681 tttgatcctt tttttgtttt ttatgtctga gctccctgca ggcatgcaag cttggcgtaa
     1741 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     1801 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     1861 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     1921 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     1981 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     2041 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     2101 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2161 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     2221 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2281 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2341 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2401 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     2461 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     2521 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     2581 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     2641 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     2701 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     2761 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     2821 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     2881 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     2941 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3001 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcggct cccacgctca
     3061 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3121 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3181 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3241 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3301 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3361 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3421 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3481 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3541 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3601 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3661 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3721 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     3781 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     3841 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa
     3901 cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc taatttttca
     3961 aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgaaag cgctatttta
     4021 ccaacgaaga atctgtgctt catttttgta aaacaaaaat gcaacgcgag agcgctaatt
     4081 tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc gagagcgcta
     4141 ttttaccaac aaagaatcta tacttctttt ttgttctaca aaaatgcatc ccgagagcgc
     4201 tatttttcta acaaagcatc ttagattact ttttttctcc tttgtgcgct ctataatgca
     4261 gtctcttgat aactttttgc actgtaggtc cgttaaggtt agaagaaggc tactttggtg
     4321 tctattttct cttccataaa aaaagcctga ctccacttcc cgcgtttact gattactagc
     4381 gaagctgcgg gtgcattttt tcaagataaa ggcatccccg attatattct ataccgatgt
     4441 ggattgcgca tactttgtga acagaaagtg atagcgttga tgattcttca ttggtcagaa
     4501 aattatgaac ggtttcttct attttgtctc tatatactac gtataggaaa tgtttacatt
     4561 ttcgtattgt tttcgattca ctctatgaat agttcttact acaatttttt tgtctaaaga
     4621 gtaatactag agataaacat aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag
     4681 cgaaaggtgg atgggtaggt tatataggga tatagcacag agatatatag caaagagata
     4741 cttttgagca atgtttgtgg aagcggtatt cgcaatattt tagtagctcg ttacagtccg
     4801 gtgcgttttt ggttttttga aagtgcgtct tcagagcgct tttggttttc aaaagcgctc
     4861 tgaagttcct atactttcta gagaatagga acttcggaat aggaacttca aagcgtttcc
     4921 gaaaacgagc gcttccgaaa atgcaacgcg agctgcgcac atacagctca ctgttcacgt
     4981 cgcacctata tctgcgtgtt gcctgtatat atatatacat gagaagaacg gcatagtgcg
     5041 tgtttatgct taaatgcgta cttatatgcg tctatttatg taggatgaaa ggtagtctag
     5101 tacctcctgt gatattatcc cattccatgc ggggtatcgt atgcttcctt cagcactacc
     5161 ctttagctgt tctatatgct gccactcctc aattggatta gtctcatcct tcaatgctat
     5221 catttccttt gatattggat catactaaga aaccattatt atcatgacat taacctataa
     5281 aaataggcgt atca
//
