LOCUS       sequence-218313-        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  JOURNAL   Unpublished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            "Unpublished"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        63..331
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        352..426
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      431..450
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     rep_origin      complement(497..1085)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(577..596)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1256..2047,2048..2116))
                     /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     1879..1898
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2117..2221)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2248..3590
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(3211..3258)
                     /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     3631..3649
                     /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 agcggatctc cacgggaccc acagtcgtag atgcgtcacg
       61 tgtctttgaa aagataatgt atgattatgc tttcactcat atttatacag aaacttgatg
      121 ttttctttcg agtatataca aggtgattac atgtacgttt gaagtacaac tctagatttt
      181 gtagtgccct cttgggctag cggtaaaggt gcgcattttt tcacacccta caatgttctg
      241 ttcaaaagat tttggtcaaa cgctgtagaa gtgaaagttg gtgcgcatgt ttcggcgttc
      301 gaaacttctc cgcagtgaaa gataaatgat cgttagtgca agctttcaaa cgttttagag
      361 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      421 tcggtggtgc tttttttgtt ttttatgtct gtttaaacaa tgtatgaaac cctgtatgga
      481 gagtgattct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
      541 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
      601 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
      661 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
      721 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
      781 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
      841 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
      901 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
      961 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     1021 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     1081 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     1141 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     1201 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     1261 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     1321 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     1381 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     1441 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     1501 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     1561 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     1621 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     1681 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     1741 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     1801 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     1861 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     1921 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     1981 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     2041 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     2101 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     2161 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     2221 gcacatttcc ccgaaaagtg ccacctgaac gaagcatctg tgcttcattt tgtagaacaa
     2281 aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa
     2341 cagaaatgca acgcgaaagc gctattttac caacgaagaa tctgtgcttc atttttgtaa
     2401 aacaaaaatg caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc tgcattttta
     2461 cagaacagaa atgcaacgcg agagcgctat tttaccaaca aagaatctat acttcttttt
     2521 tgttctacaa aaatgcatcc cgagagcgct atttttctaa caaagcatct tagattactt
     2581 tttttctcct ttgtgcgctc tataatgcag tctcttgata actttttgca ctgtaggtcc
     2641 gttaaggtta gaagaaggct actttggtgt ctattttctc ttccataaaa aaagcctgac
     2701 tccacttccc gcgtttactg attactagcg aagctgcggg tgcatttttt caagataaag
     2761 gcatccccga ttatattcta taccgatgtg gattgcgcat actttgtgaa cagaaagtga
     2821 tagcgttgat gattcttcat tggtcagaaa attatgaacg gtttcttcta ttttgtctct
     2881 atatactacg tataggaaat gtttacattt tcgtattgtt ttcgattcac tctatgaata
     2941 gttcttacta caattttttt gtctaaagag taatactaga gataaacata aaaaatgtag
     3001 aggtcgagtt tagatgcaag ttcaaggagc gaaaggtgga tgggtaggtt atatagggat
     3061 atagcacaga gatatatagc aaagagatac ttttgagcaa tgtttgtgga agcggtattc
     3121 gcaatatttt agtagctcgt tacagtccgg tgcgtttttg gttttttgaa agtgcgtctt
     3181 cagagcgctt ttggttttca aaagcgctct gaagttccta tactttctag agaataggaa
     3241 cttcggaata ggaacttcaa agcgtttccg aaaacgagcg cttccgaaaa tgcaacgcga
     3301 gctgcgcaca tacagctcac tgttcacgtc gcacctatat ctgcgtgttg cctgtatata
     3361 tatatacatg agaagaacgg catagtgcgt gtttatgctt aaatgcgtac ttatatgcgt
     3421 ctatttatgt aggatgaaag gtagtctagt acctcctgtg atattatccc attccatgcg
     3481 gggtatcgta tgcttccttc agcactaccc tttagctgtt ctatatgctg ccactcctca
     3541 attggattag tctcatcctt caatgctatc atttcctttg atattggatc atactaagaa
     3601 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc
     3661 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agcggcgcgc caacaaatat
     3721 attgcaagta gtgacctatg gtgtgtagat atacgtacag ttagttacga gcctaaagac
     3781 acaacgtgtt tgttaattat actgtcgctg taatatcttc tcttccatta tcaccggtca
     3841 ttccttgcag gggcggtagt acccggagac cctgaacttt tctttttttt ttttgcgaaa
     3901 ttaaaaagtt cattttcaat tcgacaatga ggtctacaag 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 cctccttgac agtcttgacg tgcgcagctc aggggcatga tgtgactgtc gcccgtacat
     4741 ttagcccata catccccatg tataatcatt tgcatccata cattttgatg gccgcacggc
     4801 gcgaagcaaa aattacggct cctcgctgcg gacctgcgag cagggaaacg ctcccctcac
     4861 agacgcgttg aattgtcccc acgccgcgcc cctgtagaga aatataaaag gttaggattc
     4921 gccactgagg ttcttctttc atatacttcc ttttaaaatc ttgctaggat 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
//
