LOCUS       sequence-349710-        4304 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C. auris LEUpOUT NAT marker gRNA expression construct. Use with 
            pCE35 CAS9 expression construct..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4304)
  AUTHORS   Ennis CL, Hernday AD, Nobile CJ
  TITLE     A Markerless CRISPR-Mediated System for Genome Editing in Candida 
            auris Reveals a Conserved Role for Cas5 in the Caspofungin Response.
  JOURNAL   Microbiol Spectr. 2021 Dec 22;9(3):e0182021. doi: 
            10.1128/Spectrum.01820-21. Epub 2021 Nov 3.
  PUBMED    34730409
REFERENCE   2  (bases 1 to 4304)
  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: "Microbiol 
            Spectr."; date: "2021-12-22"; volume: "9(3):e0182021. doi"; pages: "
            10.1128/Spectrum.01820-21. Epub 2021 Nov 3"
COMMENT     Sequence Label: pCE27
FEATURES             Location/Qualifiers
     source          1..4304
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(27..815)
                     /codon_start=1
                     /gene="bla(M)"
                     /product="beta-lactamase lacking the signal sequence"
                     /label=bla(M)
                     /note="allows cytosolic expression of beta-lactamase"
                     /translation="PETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMST
                     FKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMS
                     DNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATT
                     LRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAA
                     LGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
     primer_bind     650..669
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(888..992)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1060..1078
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      1649..1846
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     misc_RNA        2322..2397
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(3218..3224,3225..3242,3243..3248))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3263..3284
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3401..3418)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3572..4160)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3652..3671)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atatatgagt aaacttggtc tgacagttac caatgcttga tcagtgaggc acctatctca
       61 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
      121 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
      181 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
      241 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
      301 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
      361 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
      421 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
      481 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
      541 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
      601 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
      661 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
      721 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
      781 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
      841 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
      901 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
      961 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     1021 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     1081 tttgacggca cggccacgcg tttaaaccgc caggtgatgc tgaagctatt gaagctttgg
     1141 atggttcttt cactaccgat actgttttta gagttactgc tactggtgat ggtttcactt
     1201 tgagagaagt tccagttgat ccaccattga ctaaggtttt cccagatgat gaatccgatg
     1261 atgaatccga tgctggtgaa gatggtgatc cagattctag aactttcgtt gcttatggtg
     1321 atgatggtga tttggctggt ttcgttgttg tttcttattc tggttggaac agaagattga
     1381 ctgttgaaga tattgaagtt gctccagaac atagaggtca tggtgttggt agagctttga
     1441 tgggtttggc tactgaattc gccagagaaa gaggtgctgg tcatttgtgg ttggaagtta
     1501 ccaatgttaa tgctccagct attcatgctt atagaagaat gggtttcact ttgtgtggtt
     1561 tggatactgc tttatacgat ggtactgctt ccgatggtga acaagctttg tatatgtcta
     1621 tgccatgtcc ataataactc gagtcagtac tgacaataaa aagattcttg ttttcaagaa
     1681 cttgtcattt gtatagtttt tttatattgt agttgttcta ttttaatcaa atgttagcgt
     1741 gatttatatt ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa gtgcgcagaa
     1801 agtaatatca tgcgtcaatc gtatgtgaat gctggtcgct atactgctgt cgattcgata
     1861 ctaaagctta acttttagag tcacttaccc tacaaaggtg taagcttccc tcagatcagg
     1921 cggcacgcat ctgatatcta acatccattg taaaattagt gaagattaac actcagagcc
     1981 agttattttg aacacagatg gcttttggct gctcttatcg tctacgtttc tttttttttt
     2041 tttttttttt ttttttccta attttttacg agttcactaa ttggtttcgg gggctcaatt
     2101 ttcgaatgcg acattcataa actttgacct tttatgtgtt gatctggata attttgacct
     2161 caaaactcgg aaaaattgat gtatagcgta ttcgacatgg ctttaaaagt cgcagagttc
     2221 tacgattttc atttgcactg gcccatccgg taagcctcgg ggctccggca tgtgagggtt
     2281 cgactccctc agcagaaaac atgccgtgaa agaaaacacc agttttagag ctagaaatag
     2341 caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt
     2401 ttttctcgag tttttttatc gagtctccat tgaaggccag tgttgtcaaa ggaaccaatt
     2461 tcaccgtggt gcgtgaattg gtcggtggta tctactttgg tgagagacaa gaacaggaag
     2521 agtctgccga cggtcagagc gcttgggaca ccgagaagta cagtgtctct gaagttgaaa
     2581 gaatcaccag aatggctgcc ttcatggctt tgcagcacaa ccctcctttg cccatctggt
     2641 ccttagacaa ggccaacgtg cttgcctcat ctagattgtg gagaaagacg gtcgacaagg
     2701 tgatgagcga ggagttccct caattgacca tcaaccacca gctcattgac tctgctgcca
     2761 tgattttggt gcagagccca actaaattga acggtgttgt gatcacgtca aacatgtttg
     2821 gtgatattat ttccgacgag gcttccgtga tccctggctc cttgggtctt ttaccttctg
     2881 catctttggc ttcattacca gatacaaata aggcgtttgg tttgtacgaa ccatgccacg
     2941 gttcggctcc ggacttgcca gaaaacaagg tcaaccctgt cgccacgatt ttgtcggccg
     3001 ctatgatgtt gcgcttgtca ttagactgcc tcagagaggc agaggccttg gagaaggctg
     3061 ttcgtaacgt tttggactct ggtattcgaa ccgcagattt gaagggctca agttccactc
     3121 aggaagtggg cgatgccatt gctgctgagg taaagaagct tttgcagaaa taacggtgtt
     3181 taaaccccag cgcctggcgg gcgctcacaa ttccacacaa catacgagcc ggaagcataa
     3241 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     3301 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     3361 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     3421 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     3481 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     3541 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     3601 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     3661 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     3721 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     3781 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     3841 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     3901 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     3961 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     4021 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4081 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4141 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     4201 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     4261 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagt
//
