LOCUS       sequence-248797-        5283 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing gRNA expression cassette targeting ADE2 in 
            Saccharomyces cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5283)
  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 5283)
  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: pCfB8622
FEATURES             Location/Qualifiers
     source          1..5283
                     /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        1289..1557
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        1578..1652
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      1657..1676
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(1777..1794)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1948..2536)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2028..2047)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2707..3498,3499..3567))
                     /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     3330..3349
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3568..3672)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3699..5041
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(4662..4709)
                     /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     5082..5100
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5138..5160)
                     /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 acgccagcta
     1261 cgtgcgatag ggaacaaaag ctggagcttc tttgaaaaga taatgtatga ttatgctttc
     1321 actcatattt atacagaaac ttgatgtttt ctttcgagta tatacaaggt gattacatgt
     1381 acgtttgaag tacaactcta gattttgtag tgccctcttg ggctagcggt aaaggtgcgc
     1441 attttttcac accctacaat gttctgttca aaagattttg gtcaaacgct gtagaagtga
     1501 aagttggtgc gcatgtttcg gcgttcgaaa cttctccgca gtgaaagata aatgatcaat
     1561 tgtagagact atccacagtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     1621 tatcaacttg aaaaagtggc accgagtcgg tggtgctttt tttgtttttt atgtcttcga
     1681 gtcatgtaat tagttaatcg cgtgtttcca gctgcattaa tgaatcggcc aacgcgcggg
     1741 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     1801 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     1861 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     1921 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     1981 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     2041 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     2101 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     2161 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     2221 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     2281 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     2341 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     2401 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     2461 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     2521 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     2581 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     2641 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     2701 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     2761 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     2821 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     2881 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     2941 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     3001 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     3061 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     3121 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     3181 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     3241 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     3301 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     3361 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     3421 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     3481 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     3541 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     3601 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     3661 aggggttccg cgcacatttc cccgaaaagt gccacctgaa cgaagcatct gtgcttcatt
     3721 ttgtagaaca aaaatgcaac gcgagagcgc taatttttca aacaaagaat ctgagctgca
     3781 tttttacaga acagaaatgc aacgcgaaag cgctatttta ccaacgaaga atctgtgctt
     3841 catttttgta aaacaaaaat gcaacgcgag agcgctaatt tttcaaacaa agaatctgag
     3901 ctgcattttt acagaacaga aatgcaacgc gagagcgcta ttttaccaac aaagaatcta
     3961 tacttctttt ttgttctaca aaaatgcatc ccgagagcgc tatttttcta acaaagcatc
     4021 ttagattact ttttttctcc tttgtgcgct ctataatgca gtctcttgat aactttttgc
     4081 actgtaggtc cgttaaggtt agaagaaggc tactttggtg tctattttct cttccataaa
     4141 aaaagcctga ctccacttcc cgcgtttact gattactagc gaagctgcgg gtgcattttt
     4201 tcaagataaa ggcatccccg attatattct ataccgatgt ggattgcgca tactttgtga
     4261 acagaaagtg atagcgttga tgattcttca ttggtcagaa aattatgaac ggtttcttct
     4321 attttgtctc tatatactac gtataggaaa tgtttacatt ttcgtattgt tttcgattca
     4381 ctctatgaat agttcttact acaatttttt tgtctaaaga gtaatactag agataaacat
     4441 aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag cgaaaggtgg atgggtaggt
     4501 tatataggga tatagcacag agatatatag caaagagata cttttgagca atgtttgtgg
     4561 aagcggtatt cgcaatattt tagtagctcg ttacagtccg gtgcgttttt ggttttttga
     4621 aagtgcgtct tcagagcgct tttggttttc aaaagcgctc tgaagttcct atactttcta
     4681 gagaatagga acttcggaat aggaacttca aagcgtttcc gaaaacgagc gcttccgaaa
     4741 atgcaacgcg agctgcgcac atacagctca ctgttcacgt cgcacctata tctgcgtgtt
     4801 gcctgtatat atatatacat gagaagaacg gcatagtgcg tgtttatgct taaatgcgta
     4861 cttatatgcg tctatttatg taggatgaaa ggtagtctag tacctcctgt gatattatcc
     4921 cattccatgc ggggtatcgt atgcttcctt cagcactacc ctttagctgt tctatatgct
     4981 gccactcctc aattggatta gtctcatcct tcaatgctat catttccttt gatattggat
     5041 catactaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     5101 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     5161 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     5221 cagcgggctg catttaaatg atctgtttag cttgcctcgt ccccgccggg tcacccggcc
     5281 agc
//
