LOCUS       sequence-210455-        6244 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses gRNA to target Nte1 locus and has HygR marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6244)
  TITLE     Plasmids for Cas9-mediated genome editing in S. cerevisiae with 
            dominant markers
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6244)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCB32
FEATURES             Location/Qualifiers
     source          1..6244
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        64..332
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        353..427
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      432..451
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     rep_origin      complement(498..1086)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(578..597)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1257..2048,2049..2117))
                     /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     1880..1899
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2118..2222)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2249..3591
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(3212..3259)
                     /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     3632..3650
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     gene            4661..6236
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        4661..5004
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             5005..6033
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             5005..6033
                     /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"
     terminator      6039..6236
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
ORIGIN
        1 gacggtcaca gcttgtctgt aagcggatct ccacgggacc cacagtcgta gatgcgtcac
       61 gtgtctttga aaagataatg tatgattatg ctttcactca tatttataca gaaacttgat
      121 gttttctttc gagtatatac aaggtgatta catgtacgtt tgaagtacaa ctctagattt
      181 tgtagtgccc tcttgggcta gcggtaaagg tgcgcatttt ttcacaccct acaatgttct
      241 gttcaaaaga ttttggtcaa acgctgtaga agtgaaagtt ggtgcgcatg tttcggcgtt
      301 cgaaacttct ccgcagtgaa agataaatga tcgttagtgc aagctttcaa acgttttaga
      361 gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
      421 gtcggtggtg ctttttttgt tttttatgtc tgtttaaaca atgtatgaaa ccctgtatgg
      481 agagtgattc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
      541 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
      601 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
      661 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
      721 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
      781 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
      841 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
      901 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
      961 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     1021 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     1081 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     1141 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     1201 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     1261 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     1321 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     1381 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     1441 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     1501 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     1561 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     1621 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     1681 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     1741 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     1801 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     1861 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     1921 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     1981 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     2041 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     2101 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     2161 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     2221 cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt ttgtagaaca
     2281 aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca tttttacaga
     2341 acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt catttttgta
     2401 aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag ctgcattttt
     2461 acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta tacttctttt
     2521 ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc ttagattact
     2581 ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc actgtaggtc
     2641 cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa aaaagcctga
     2701 ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt tcaagataaa
     2761 ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga acagaaagtg
     2821 atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct attttgtctc
     2881 tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca ctctatgaat
     2941 agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat aaaaaatgta
     3001 gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt tatataggga
     3061 tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg aagcggtatt
     3121 cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga aagtgcgtct
     3181 tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta gagaatagga
     3241 acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa atgcaacgcg
     3301 agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt gcctgtatat
     3361 atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta cttatatgcg
     3421 tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc cattccatgc
     3481 ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct gccactcctc
     3541 aattggatta gtctcatcct tcaatgctat catttccttt gatattggat catactaaga
     3601 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct
     3661 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagcggcgcg ccaacaaata
     3721 tattgcaagt agtgacctat ggtgtgtaga tatacgtaca gttagttacg agcctaaaga
     3781 cacaacgtgt ttgttaatta tactgtcgct gtaatatctt ctcttccatt atcaccggtc
     3841 attccttgca ggggcggtag tacccggaga ccctgaactt ttcttttttt ttttgcgaaa
     3901 ttaaaaagtt cattttcaat tcgacaatga gatctacaag ccattgtttt atgttgatga
     3961 gagccagctt aaagagttaa aaatttcata gctaatggtg acagggggaa tggcaagcaa
     4021 gtgggatcag aagggtatgg acattgccta tgaggaggcg gccttaggtt acaaagaggg
     4081 tggtgttcct attggcggat gtcttatcaa taacaaagac ggaagtgttc tcggtcgtgg
     4141 tcacaacatg agatttcaaa agggatccgc cacactacat ggtgagatct ccactttgga
     4201 aaactgtggg agattagagg gcaaagtgta caaagatacc actttgtata cgacgctgtc
     4261 tccatgcgac atgtgtacag gtgccatcat catgtatggt attccacgct gtgttgtcgg
     4321 tgagaacgtt aatttcaaaa gtaagggcga gaaatattta caaactagag gtcacgaggt
     4381 tgttgttgtt gacgatgaga ggtgtaaaaa gatcatgaaa caatttatcg atgaaagacc
     4441 tcaggattgg tttgaagata ttggtgagta gagcacgcag cacgctgtat ttacgtattt
     4501 aattttatat atttgtgcat acactactag ggaagacttg aaaaaaacct aggaaatgaa
     4561 aaaacgacac aggaagtccc gtatttacta ttttttcctt ccttttgatg gggcagggcg
     4621 gaaatagagg ataggataag ccattaccct gttatcccta gacatggagg cccagaatac
     4681 cctccttaac agtcttgacg tgcgcagctc aggggcatga tgtgactgtc gcccgtacat
     4741 ttagcccata catccccatg tataatcatt tgcatccata cattttgatg gccgcacggc
     4801 gcgaagcaaa aattacggct cctcgctgca gacctgcgag cagggaaacg ctcccctcac
     4861 agacgcgttg aattgtcccc acgccgcgcc cctgtagaga aatataaaag gttaggattt
     4921 gccactgagg ttcttctttc atgtacttcc ttttaaaatc ttgctagggt acagttctca
     4981 catcacatcc gaacataaac aaccatgggt aaaaagcctg aactcaccgc gacgtctgtc
     5041 gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc
     5101 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat
     5161 agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg
     5221 ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc
     5281 tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt
     5341 ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc
     5401 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata
     5461 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt
     5521 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc
     5581 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata
     5641 acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac
     5701 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg
     5761 aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt
     5821 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt
     5881 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc
     5941 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga
     6001 cgccccagca ctcgtccgag ggcaaaggaa taatcagtac tgacaataaa aagattcttg
     6061 ttttcaagaa cttgtcattt gtatagtttt tttatattgt agttgttcta ttttaatcaa
     6121 atgttagcgt gatttatatt ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa
     6181 gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat gctggtcgct atactgttaa
     6241 ttaa
//
