LOCUS       sequence-480869-        5740 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of DNA into Cryptococcus neoformans Safe Haven 1 locus, 
            contains BLE marker for selection on phleomycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5740)
  AUTHORS   Boucher MJ, Madhani HD
  TITLE     CRISPR/Cas9-compatible plasmids enabling seven dominant genetic 
            selection methods for the human fungal pathogen Cryptococcus 
            neoformans.
  JOURNAL   Microbiol Spectr. 2025 Sep 25:e0193525. doi: 
            10.1128/spectrum.01935-25.
  PUBMED    40996288
REFERENCE   2  (bases 1 to 5740)
  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. 2025 Sep 25:e0193525. doi: 10.1128/spectrum.01935-25."
COMMENT     Sequence Label: pBHM2672
FEATURES             Location/Qualifiers
     source          1..5740
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      2239..2248
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(3216..3581)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWKL"
     primer_bind     3350..3372
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3364..3381
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3365..3381
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3391..3410
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3391..3409
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    3418..3525
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     3434..3450
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     3435..3451
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3485..3501)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3485..3501)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3538..3558)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3538..3556)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3577..3593)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3577..3593)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3590..3612)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3601..3617
                     /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)."
     promoter        complement(join(3625..3631,3632..3649,3650..3655))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3670..3691
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3808..3825)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3979..4567)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4059..4078)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4738..5529,5530..5598))
                     /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     5361..5380
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5599..5703)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agcggcaaga tctgcccata gacttcaaat catggaattg gacgtttatc ttcttgcttt
       61 ggatatccac cctatcgtac aatcttggca ctttggatca tacgaggcac tttgcggact
      121 agggaatggt tatcatcaat ttcttggacc tctccatgac acctggcatc gtcgagctac
      181 cacagccccc tgaattctta aaggcgaacc ccccttccta ggaaccttga gcgctggact
      241 ggatggcact ctgagaccgc ctatgcttaa ggacgcgagc ttacccttcc attgatggaa
      301 gtcgatcatt tatgataata gaacacggcg gaacttgact ttacaggcca agggaagcaa
      361 caggagaaga agctaaggtt ataacaggct cagcatattt gtatatacgg tgccattcaa
      421 cgctgaacac tttgtactca cggtctgccc gttgaataac accatctgct gatgatcatc
      481 ccaatttgtg agtcttattt gccattgatc ctggtgtata accgatactg aagcccttta
      541 ggtctgatca gttatagacg tggtatattc attcttccta gattagctat gccgaaagag
      601 atggatacct tgatgtgact ggaatgccat gaagcaggag attgtagcag ctcaaattgg
      661 agaaggatgt tgcaaccgtc ggccgtgaag gtcttgtcat atctttgtct gcctgggaga
      721 atatgtacaa gtgccccgat gttactgatg gaaaacatcg tgtggcgcgc cacgatactt
      781 gtgttaatta aaccaaacca gtctgtgcca agtggattgc ttatgcatcg agtggcgagc
      841 ccactgacct ctgggcccct tttttgaact ataaaaagcg gccaagtctg atttcggtct
      901 cacaatcatc aacatcgact tctcttttcg cctcgatcaa cctaaacaac ggtcactcag
      961 ccatgaactg gacaatggaa tatttcgcct tgccttggat ctttggcctc tagactcaat
     1021 caacgtcagg ataaaggtga gcataggttg ctcactcact gtagcctttt tttcccttct
     1081 tcttggtcca gatggttccc aaggctctat aaagtgtatc tgtcagttcc acttccttac
     1141 tccgaagcga ttcagagttg taaagtgcgt ctgaccccct gattatatct caaagtccct
     1201 tcacaaattc agacatgatg cgataaagtg ccggattgtc attcgcgtct tgtgactgta
     1261 tagaaagtgc cggcatcctc ttttcttttc ctctttggcc gagcgactca gagttgtgaa
     1321 gcctgctggt atctgtctgt tcccttgccc tgataaagaa tatcgaagag catttctttg
     1381 attcacaata cgttgacgaa tggtgtgatg gatcctaagc agcgccagcc ctttgaactt
     1441 gtgtcgaaaa aaaaaaaaaa acagcatatg atacgtaaag ggaagaaaag gatggagctg
     1501 actgttttga caggcataat tttgagcgtg cttcattggc cggtttatct gtattaacac
     1561 ggaagagatg tagaaactag cttcctggtt tcagagacaa ggagccatga agatcctgag
     1621 gattcaacgg cgttgctgag ggtgggaagg atggtattgt tgtctgtaga tagattaatg
     1681 ggcatataat gtcaaaaaag atgattgaca cgatgtctgt ctcgaattcg cagtgtgcta
     1741 aaattgtatc acttctctaa tatgtatata tacaccctct aaggaaaatt aaccccttac
     1801 cgccttcacg aattcttagt cctgctcctc ggcgacgaag tggacacagt taccggcggg
     1861 gtctcggagg gcgaactctc gaccccaggg ctgctcaccg atctcggtca tggcgggacc
     1921 ggaggcgtct cggaagttgg tggagacgac ctcggaccac tcggcgtaga gctcgtcgag
     1981 acctcggacc cagacccagg cgagggtgtt gtcggggacg acctggtcct ggacggcgga
     2041 gatgaagagg gtgacgtcgt ctcgctgtcg ggagatgtta gtggagtaac atgttgcggg
     2101 aaacggggtt acatacgacg acaccggcga agtcgtcctc gacgaagtct cgggagaaac
     2161 cgagtcggtc ggtccagaac tcgacggcac cggcgacgtc tcgggcggtg aggacgggga
     2221 cggcggaggt gagcttggcc atggcggccg ccatagacat gttgggcgag tttactaatg
     2281 gaaaaagaca attagcaaag aggctgcata ttaggagacg cggtgaagat tggggaggac
     2341 aatagggtcc gtgcaccaaa aagtcctgac ggcagctctg ttcaacgcac atccatctct
     2401 tccagctatg cgtgtggcac cagcacggat ggattgttgc accaaaagga caaggcgaga
     2461 tggatgcagg tcgaggcgat gcggtgctat cggccctcgt tgtcgaacgc cacgtcgtcc
     2521 tctcggcgac cgtcaatcct ggaaggaaat gaccaagcgt gcacccagca tccagcatcc
     2581 aacatccagc acccagcacc tagcacccat gatccaatgg ccggcatgca gatcagcgtg
     2641 gggagagcac catctagcag cccctggcag gcccagcatt gtgctcggat gtgcgatagg
     2701 cggtgggtgc gataaggtgg attagtggag gatggaggac tgagtggcgg gcaagggaag
     2761 ggggcgggaa gatgactcac cttgggcagt gggtggggga tgcggtatag gggatgtatg
     2821 tggatgctgg cggaggatag aagctgtaag ctgagctggt gccaacggtg acgctgtgag
     2881 agtggtctcg gcgaccctga cggcgactcg gcgttacccc gtgtggcaca ccggacgcgc
     2941 gtcgagtgac gttgctgcta ttgttgttac catcatcctc tcctccgaca accatacact
     3001 cagtgcacac catgccactc gaatcctgca tgcttatgtg agtcctcccc ttcccgtgcg
     3061 ccgctctcca gctcacatcc tcgcagccat ggactcaacc ctatctcggt ctattctttt
     3121 gatttataag ggattttgcc gatttcggcc tattggttaa aaaaatgagc tgatttaaca
     3181 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttccattc gccattcagg
     3241 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg
     3301 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
     3361 cgttgtaaaa cgacggccag tgagcgcgcg taatacgact cactataggg cgaattgggt
     3421 accgggcccc ccctcgaggt cgacggtatc gataagcttg atatcgaatt cctgcagccc
     3481 gggggatcca ctagttctag agcggccgcc accgcggtgg agctccagct tttgttccct
     3541 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     3601 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     3661 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     3721 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3781 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3841 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3901 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3961 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4021 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4081 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4141 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4201 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4261 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4321 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4381 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     4441 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4501 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4561 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4621 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4681 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4741 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4801 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4861 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     4921 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     4981 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5041 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5101 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5161 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5221 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5281 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5341 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5401 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5461 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5521 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5581 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5641 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5701 gcgcacattt ccccgaaaag tgccacctga cgcgccctgt
//
