LOCUS       sequence-248788-        3130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for PCR amplification of ADE2 gRNA biobrick for targeting 
            Saccharomyces cerevisiae ADE2 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3130)
  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 3130)
  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: pCfB8792
FEATURES             Location/Qualifiers
     source          1..3130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    89..110
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(125..130,131..148,149..155)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    163..179
                     /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)."
     primer_bind     168..190
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     187..203
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     187..203
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        282..550
                     /label=SNR52 promoter
                     /note="promoter for the S. cerevisiae small nucleolar RNA 
                     gene SNR52"
     misc_RNA        571..645
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      650..669
                     /label=SUP4 terminator
                     /note="transcription terminator for the S. cerevisiae SUP4 
                     tRNA gene"
     primer_bind     complement(717..734)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(717..733)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(726..748)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(942..961)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1061..1083
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1121..1139)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1207..1311
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1312..1380,1381..2172)
                     /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     complement(1530..1549)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2343..2931
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2832..2851
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3085..3102
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
       61 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
      121 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
      181 ttcacacagg aaacagctat gaccatgatt acgccaagct tgcatgcctg caggtcgact
      241 ctagaggatc ccccgtgcga tagggaacaa aagctggagc ttctttgaaa agataatgta
      301 tgattatgct ttcactcata tttatacaga aacttgatgt tttctttcga gtatatacaa
      361 ggtgattaca tgtacgtttg aagtacaact ctagattttg tagtgccctc ttgggctagc
      421 ggtaaaggtg cgcatttttt cacaccctac aatgttctgt tcaaaagatt ttggtcaaac
      481 gctgtagaag tgaaagttgg tgcgcatgtt tcggcgttcg aaacttctcc gcagtgaaag
      541 ataaatgatc aattgtagag actatccaca gttttagagc tagaaatagc aagttaaaat
      601 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtggtgct ttttttgttt
      661 tttatgtctt cgagtcatgt aattagttaa tcgcgtgggg taccgagctc gaattcactg
      721 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
      781 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
      841 tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg
      901 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc
      961 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     1021 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     1081 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     1141 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     1201 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1261 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     1321 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     1381 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     1441 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     1501 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     1561 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     1621 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     1681 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     1741 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     1801 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     1861 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     1921 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     1981 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     2041 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     2101 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     2161 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     2221 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     2281 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2341 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2401 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     2461 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     2521 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     2581 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     2641 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     2701 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     2761 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     2821 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     2881 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     2941 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     3001 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     3061 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     3121 atacgcaaac
//
