LOCUS       sequence-269842-        5339 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Helper plasmid for SaCas9 gRNA cloning and expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5339)
  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 5339)
  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*-SaSgH
FEATURES             Location/Qualifiers
     source          1..5339
                     /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        194..409
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             410..1213
                     /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        1302..1570
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     terminator      1719..1738
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(1776..1792)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1776..1792)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1789..1811)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1800..1816
                     /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(1824..1830,1831..1848,1849..1854))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1869..1890
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2007..2024)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2178..2766)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2258..2277)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2937..3728,3729..3797))
                     /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     3560..3579
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3798..3902)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3929..5271
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(4892..4939)
                     /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     5312..5330
                     /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 cttttcaatt catcattttt tttttattct tttttttgat ttcggtttcc
      241 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag
      301 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt
      361 cttaacccaa ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc
      421 tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc tatttaatat
      481 catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt
      541 actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac atgtggatat
      601 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta
      661 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca
      721 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt
      781 ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc
      841 tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta ctggagaata
      901 tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc
      961 tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga cacccggtgt
     1021 gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtcgt
     1081 ttctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg gaagggatgc
     1141 taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga gaagatgcgg
     1201 ccagcaaaac taaaaaactg tattataagt aaatgcatgt atactaaact cacaaattag
     1261 agcttcaatt taattatatc agttattacc ctgtgctcga gtctttgaaa agataatgta
     1321 tgattatgct ttcactcata tttatacaga aacttgatgt tttctttcga gtatatacaa
     1381 ggtgattaca tgtacgtttg aagtacaact ctagattttg tagtgccctc ttgggctagc
     1441 ggtaaaggtg cgcatttttt cacaccctac aatgttctgt tcaaaagatt ttggtcaaac
     1501 gctgtagaag tgaaagttgg tgcgcatgtt tcggcgttcg aaacttctcc gcagtgaaag
     1561 ataaatgatc ggagaccgag gtctccgttt tagtactctg taattttagg tatgaggtag
     1621 acgaaaattg tacttatacc taaaattaca gaatctacta aaacaaggca aaatgccgtg
     1681 tttatctcgt caacttgttg gcgagatttt tttgatcctt tttttgtttt ttatgtctga
     1741 gctccctgca ggcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     1801 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     1861 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     1921 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     1981 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     2041 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     2101 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     2161 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     2221 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2281 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2341 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     2401 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2461 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2521 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     2581 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     2641 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     2701 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     2761 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2821 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     2881 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2941 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3001 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     3061 gctgcaatga taccgcggct cccacgctca ccggctccag atttatcagc aataaaccag
     3121 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     3181 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3241 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3301 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3361 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3421 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3481 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     3541 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     3601 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     3661 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     3721 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     3781 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     3841 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     3901 cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt ttgtagaaca
     3961 aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca tttttacaga
     4021 acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt catttttgta
     4081 aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag ctgcattttt
     4141 acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta tacttctttt
     4201 ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc ttagattact
     4261 ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc actgtaggtc
     4321 cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa aaaagcctga
     4381 ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt tcaagataaa
     4441 ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga acagaaagtg
     4501 atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct attttgtctc
     4561 tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca ctctatgaat
     4621 agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat aaaaaatgta
     4681 gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt tatataggga
     4741 tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg aagcggtatt
     4801 cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga aagtgcgtct
     4861 tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta gagaatagga
     4921 acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa atgcaacgcg
     4981 agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt gcctgtatat
     5041 atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta cttatatgcg
     5101 tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc cattccatgc
     5161 ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct gccactcctc
     5221 aattggatta gtctcatcct tcaatgctat catttccttt gatattggat catactaaga
     5281 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc
//
