LOCUS       sequence-248798-        5726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing gRNA expression cassettes targeting the X-4 and 
            XII-5 integration sites in Saccharomyces cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5726)
  AUTHORS   Milne N, Tramontin LRR, Borodina I
  TITLE     A teaching protocol demonstrating the use of EasyClone and 
            CRISPR/Cas9 for metabolic engineering of Saccharomyces cerevisiae 
            and Yarrowia lipolytica.
  JOURNAL   FEMS Yeast Res. 2019 Sep 26. pii: foz062. doi: 
            10.1093/femsyr/foz062.
  PUBMED    31556952
REFERENCE   2  (bases 1 to 5726)
  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: "FEMS Yeast 
            Res. 2019 Sep 26. pii: foz062. doi: 10.1093/femsyr/foz062."
COMMENT     Sequence Label: pCfB5191
FEATURES             Location/Qualifiers
     source          1..5726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     gene            1..1120
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        1..344
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             345..917
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTTTGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      923..1120
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        1294..1562
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        1583..1657
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1662..1681
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     promoter        1731..1999
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        2020..2094
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      2099..2118
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(2220..2237)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2391..2979)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2471..2490)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3150..3941,3942..4010))
                     /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     3773..3792
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4011..4115)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4142..5484
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(5105..5152)
                     /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     5525..5543
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5581..5603)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacatggagg cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga
       61 tgtgactgtc gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata
      121 cattttgatg gccgcacggc gcgaagcaaa aattacggct cctcgctgca gacctgcgag
      181 cagggaaacg ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga
      241 aatataaaag gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc
      301 ttgctaggat acagttctca catcacatcc gaacataaac aaccatgggt accactcttg
      361 acgacacggc ttaccggtac cgcaccagtg tcccggggga cgccgaggcc atcgaggcac
      421 tggatgggtc cttcaccacc gacaccgttt tccgcgtcac caccaccggg gacggcttca
      481 ccctgcggga ggtgccggtg gacccgcccc tgaccaaggt gttccccgac gacgaatcgg
      541 acgacgaatc ggacgacggg gaggacggcg acccggactc ccggacgttc gtcgcgtacg
      601 gggacgacgg cgacctggcg ggcttcgtgg tcgtctcgta ctccggctgg aaccgccggc
      661 tgaccgtcga ggacatcgag gtcgccccgg agcaccgggg gcacggggtc gggcgcgcgt
      721 tgatggggct cgcgacggag ttcgcccgcg agcggggcgc cgggcacctc tggctggagg
      781 tcaccaacgt caacgcaccg gcgatccacg cgtaccggcg gatggggttc accctctgcg
      841 gcctggacac cgccctgtac gacggcaccg cctcggacgg cgagcaggcg ctctacatga
      901 gcatgccctg cccctaatca gtactgacaa taaaaagatt cttgttttca agaacttgtc
      961 atttgtatag tttttttata ttgtagttgt tctattttaa tcaaatgtta gcgtgattta
     1021 tatttttttt cgcctcgaca tcatctgccc agatgcgaag ttaagtgcgc agaaagtaat
     1081 atcatgcgtc aatcgtatgt gaatgctggt cgctatactg ctgtcgattc gatactaacg
     1141 ccgccatcca gtgtcgaaaa cgagctgcgg ccgcctacag ggcgcgtcca ttcgccattc
     1201 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
     1261 gaatgcgtgc gatagggaac aaaagctgga gcttctttga aaagataatg tatgattatg
     1321 ctttcactca tatttataca gaaacttgat gttttctttc gagtatatac aaggtgatta
     1381 catgtacgtt tgaagtacaa ctctagattt tgtagtgccc tcttgggcta gcggtaaagg
     1441 tgcgcatttt ttcacaccct acaatgttct gttcaaaaga ttttggtcaa acgctgtata
     1501 agtgaaagtt ggtgcgcatg tttcggcgtt cgaaacttct ccgcagtgaa agataaatga
     1561 tccgccattc aagagtagca acgttttaga gctagaaata gcaagttaaa ataaggctag
     1621 tccgttatca acttgaaaaa gtggcaccga gtcggtggtg ctttttttgt tttttatgtc
     1681 ttcgagtcat gtaattagtt aagtgcaggt agggaacaaa agctggagct tctttgaaaa
     1741 gataatgtat gattatgctt tcactcatat ttatacagaa acttgatgtt ttctttcgag
     1801 tatatacaag gtgattacat gtacgtttga agtacaactc tagattttgt agtgccctct
     1861 tgggctagcg gtaaaggtgc gcattttttc acaccctaca atgttctgtt caaaagattt
     1921 tggtcaaacg ctgtagaagt gaaagttggt gcgcatgttt cggcgttcga aacttctccg
     1981 cagtgaaaga taaatgatct tgtcacagtg tcacatcagg ttttagagct agaaatagca
     2041 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtggtgctt
     2101 tttttgtttt ttatgtcttc gagtcatgta attagttaat cgcgtgcatt ccagctgcat
     2161 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     2221 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     2281 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     2341 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2401 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2461 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2521 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     2581 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     2641 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     2701 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     2761 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     2821 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     2881 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     2941 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3001 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     3061 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3121 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3181 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     3241 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     3301 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3361 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3421 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3481 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3541 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     3601 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     3661 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     3721 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     3781 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     3841 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     3901 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     3961 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4021 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4081 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4141 gaacgaagca tctgtgcttc attttgtaga acaaaaatgc aacgcgagag cgctaatttt
     4201 tcaaacaaag aatctgagct gcatttttac agaacagaaa tgcaacgcga aagcgctatt
     4261 ttaccaacga agaatctgtg cttcattttt gtaaaacaaa aatgcaacgc gagagcgcta
     4321 atttttcaaa caaagaatct gagctgcatt tttacagaac agaaatgcaa cgcgagagcg
     4381 ctattttacc aacaaagaat ctatacttct tttttgttct acaaaaatgc atcccgagag
     4441 cgctattttt ctaacaaagc atcttagatt actttttttc tcctttgtgc gctctataat
     4501 gcagtctctt gataactttt tgcactgtag gtccgttaag gttagaagaa ggctactttg
     4561 gtgtctattt tctcttccat aaaaaaagcc tgactccact tcccgcgttt actgattact
     4621 agcgaagctg cgggtgcatt ttttcaagat aaaggcatcc ccgattatat tctataccga
     4681 tgtggattgc gcatactttg tgaacagaaa gtgatagcgt tgatgattct tcattggtca
     4741 gaaaattatg aacggtttct tctattttgt ctctatatac tacgtatagg aaatgtttac
     4801 attttcgtat tgttttcgat tcactctatg aatagttctt actacaattt ttttgtctaa
     4861 agagtaatac tagagataaa cataaaaaat gtagaggtcg agtttagatg caagttcaag
     4921 gagcgaaagg tggatgggta ggttatatag ggatatagca cagagatata tagcaaagag
     4981 atacttttga gcaatgtttg tggaagcggt attcgcaata ttttagtagc tcgttacagt
     5041 ccggtgcgtt tttggttttt tgaaagtgcg tcttcagagc gcttttggtt ttcaaaagcg
     5101 ctctgaagtt cctatacttt ctagagaata ggaacttcgg aataggaact tcaaagcgtt
     5161 tccgaaaacg agcgcttccg aaaatgcaac gcgagctgcg cacatacagc tcactgttca
     5221 cgtcgcacct atatctgcgt gttgcctgta tatatatata catgagaaga acggcatagt
     5281 gcgtgtttat gcttaaatgc gtacttatat gcgtctattt atgtaggatg aaaggtagtc
     5341 tagtacctcc tgtgatatta tcccattcca tgcggggtat cgtatgcttc cttcagcact
     5401 accctttagc tgttctatat gctgccactc ctcaattgga ttagtctcat ccttcaatgc
     5461 tatcatttcc tttgatattg gatcatacta agaaaccatt attatcatga cattaaccta
     5521 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     5581 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     5641 cagacaagcc cgtcagggcg cgtcagcggg ctgcatttaa atgatctgtt tagcttgcct
     5701 cgtccccgcc gggtcacccg gccagc
//
