LOCUS       sequence-494489-        6504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ura3 deletion gRNA cassette carried by pRS42H.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6504)
  AUTHORS   Zhang GC, Kong II, Kim H, Liu JJ, Cate JH, Jin YS
  TITLE     Construction of a quadruple auxotrophic mutant of an industrial 
            polyploid saccharomyces cerevisiae strain by using RNA-guided Cas9 
            nuclease.
  JOURNAL   Appl Environ Microbiol. 2014 Dec;80(24):7694-701. doi: 
            10.1128/AEM.02310-14. Epub 2014 Oct 3.
  PUBMED    25281382
REFERENCE   2  (bases 1 to 6504)
  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"; doi: 
            "10.1128/AEM.02310-14"; journalName: "Appl Environ Microbiol"; date:
            "2014-12"; volume: "80"; issue: "24"; pages: "7694-701"
COMMENT     Sequence Label: gRNA-ura-HYB
FEATURES             Location/Qualifiers
     source          1..6504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             348..1376
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             348..1376
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     primer_bind     complement(1400..1418)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(1662..2117)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1799..1820)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2011..2030
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2243..2265
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2257..2274
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2258..2274
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2284..2303
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2284..2302
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(2317..2336)
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     misc_RNA        complement(2341..2415)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2436..2704)
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     primer_bind     complement(2723..2743)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2723..2741)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2762..2778)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2762..2778)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2775..2797)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2786..2802
                     /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(2810..2816,2817..2834,2835..2840))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2855..2876
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2993..3010)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3164..3752)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3244..3263)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3923..4714,4715..4783))
                     /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     4546..4565
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4784..4888)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4915..6257
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5878..5925)
                     /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     6298..6316
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6354..6376)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6476..6495
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cagcgacatg gggcccagaa taccctcctt gacagtcttg acgtgcgcag ctcaggggca
       61 tgatgtgact gtcgcccgta catttagccc atacatcccc atgtataatc atttgcatcc
      121 atacattttg atggccgcac ggcgcgaagc aaaaattacg gctcctcgct gcggacctgc
      181 gagcagggaa acgctcccct cacagacgcg ttgaattgtc cccacgccgc gcccctgtag
      241 agaaatataa aaggttagga tttgccactg aggttcttct ttcatatact tccttttaaa
      301 atcttgctag gatacagttc tcacatcaca tccgaacata aacaaccatg ggtaaaaagc
      361 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
      421 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
      481 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
      541 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca
      601 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
      661 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg
      721 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
      781 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
      841 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
      901 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
      961 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
     1021 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
     1081 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg
     1141 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     1201 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     1261 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     1321 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaataatctc
     1381 gagtcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa
     1441 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
     1501 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
     1561 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cggcggtgtg
     1621 aaataccgca cagatgcgta aggagaaaat accgcatcag gaaattgtaa gcgttaatat
     1681 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     1741 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     1801 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     1861 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     1921 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     1981 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     2041 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     2101 gccgctacag ggcgcgtcca ttcgccattc aggctgcgca actgttggga agggcgatcg
     2161 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     2221 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc
     2281 gcgtaatacg actcactata gggcgaattg ggtaccagac ataaaaaaca aaaaaagcac
     2341 caccgactcg gtgccacttt ttcaagttga taacggacta gccttatttt aacttgctat
     2401 ttctagctct aaaacccaag tacaattttt tactcgatca tttatctttc actgcggaga
     2461 agtttcgaac gccgaaacat gcgcaccaac tttcacttct acagcgtttg accaaaatct
     2521 tttgaacaga acattgtagg gtgtgaaaaa atgcgcacct ttaccgctag cccaagaggg
     2581 cactacaaaa tctagagttg tacttcaaac gtacatgtaa tcaccttgta tatactcgaa
     2641 agaaaacatc aagtttctgt ataaatatga gtgaaagcat aatcatacat tatcttttca
     2701 aagagagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat
     2761 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     2821 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     2881 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     2941 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     3001 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3061 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3121 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3181 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3241 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3301 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3361 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3421 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3481 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3541 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3601 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3661 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3721 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3781 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     3841 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     3901 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     3961 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4021 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4081 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4141 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4201 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4261 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4321 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4381 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4441 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4501 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4561 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     4621 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     4681 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4741 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4801 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     4861 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgaacgaa
     4921 gcatctgtgc ttcattttgt agaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca
     4981 aagaatctga gctgcatttt tacagaacag aaatgcaacg cgaaagcgct attttaccaa
     5041 cgaagaatct gtgcttcatt tttgtaaaac aaaaatgcaa cgcgagagcg ctaatttttc
     5101 aaacaaagaa tctgagctgc atttttacag aacagaaatg caacgcgaga gcgctatttt
     5161 accaacaaag aatctatact tcttttttgt tctacaaaaa tgcatcccga gagcgctatt
     5221 tttctaacaa agcatcttag attacttttt ttctcctttg tgcgctctat aatgcagtct
     5281 cttgataact ttttgcactg taggtccgtt aaggttagaa gaaggctact ttggtgtcta
     5341 ttttctcttc cataaaaaaa gcctgactcc acttcccgcg tttactgatt actagcgaag
     5401 ctgcgggtgc attttttcaa gataaaggca tccccgatta tattctatac cgatgtggat
     5461 tgcgcatact ttgtgaacag aaagtgatag cgttgatgat tcttcattgg tcagaaaatt
     5521 atgaacggtt tcttctattt tgtctctata tactacgtat aggaaatgtt tacattttcg
     5581 tattgttttc gattcactct atgaatagtt cttactacaa tttttttgtc taaagagtaa
     5641 tactagagat aaacataaaa aatgtagagg tcgagtttag atgcaagttc aaggagcgaa
     5701 aggtggatgg gtaggttata tagggatata gcacagagat atatagcaaa gagatacttt
     5761 tgagcaatgt ttgtggaagc ggtattcgca atattttagt agctcgttac agtccggtgc
     5821 gtttttggtt ttttgaaagt gcgtcttcag agcgcttttg gttttcaaaa gcgctctgaa
     5881 gttcctatac tttctagaga ataggaactt cggaatagga acttcaaagc gtttccgaaa
     5941 acgagcgctt ccgaaaatgc aacgcgagct gcgcacatac agctcactgt tcacgtcgca
     6001 cctatatctg cgtgttgcct gtatatatat atacatgaga agaacggcat agtgcgtgtt
     6061 tatgcttaaa tgcgtactta tatgcgtcta tttatgtagg atgaaaggta gtctagtacc
     6121 tcctgtgata ttatcccatt ccatgcgggg tatcgtatgc ttccttcagc actacccttt
     6181 agctgttcta tatgctgcca ctcctcaatt ggattagtct catccttcaa tgctatcatt
     6241 tcctttgata ttggatcata ctaagaaacc attattatca tgacattaac ctataaaaat
     6301 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
     6361 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     6421 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
     6481 tcagagcaga ttgtactgag agtg
//
