LOCUS       sequence-480871-        5764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of DNA into Cryptococcus neoformans Safe Haven 1 locus, 
            contains BSD marker for selection on blasticidin S.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5764)
  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 5764)
  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: pBHM2673
FEATURES             Location/Qualifiers
     source          1..5764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      2256..2265
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(3233..3598)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWSSTAVAAALELVDPPGCRNSISSLSI
                     PSTSRGGPVPNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWKL"
     primer_bind     3367..3389
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3381..3398
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3382..3398
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3408..3427
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3408..3426
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    3435..3542
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     3451..3467
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     3452..3468
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3502..3518)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3502..3518)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3555..3575)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3555..3573)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3594..3610)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3594..3610)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3607..3629)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3618..3634
                     /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(3642..3648,3649..3666,3667..3672))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3687..3708
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3825..3842)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3996..4584)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4076..4095)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4755..5546,5547..5615))
                     /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     5378..5397
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5616..5720)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agatctgccc atagacttca aatcatggaa ttggacgttt atcttcttgc tttggatatc
       61 caccctatcg tacaatcttg gcactttgga tcatacgagg cactttgcgg actagggaat
      121 ggttatcatc aatttcttgg acctctccat gacacctggc atcgtcgagc taccacagcc
      181 ccctgaattc ttaaaggcga accccccttc ctaggaacct tgagcgctgg actggatggc
      241 actctgagac cgcctatgct taaggacgcg agcttaccct tccattgatg gaagtcgatc
      301 atttatgata atagaacacg gcggaacttg actttacagg ccaagggaag caacaggaga
      361 agaagctaag gttataacag gctcagcata tttgtatata cggtgccatt caacgctgaa
      421 cactttgtac tcacggtctg cccgttgaat aacaccatct gctgatgatc atcccaattt
      481 gtgagtctta tttgccattg atcctggtgt ataaccgata ctgaagccct ttaggtctga
      541 tcagttatag acgtggtata ttcattcttc ctagattagc tatgccgaaa gagatggata
      601 ccttgatgtg actggaatgc catgaagcag gagattgtag cagctcaaat tggagaagga
      661 tgttgcaacc gtcggccgtg aaggtcttgt catatctttg tctgcctggg agaatatgta
      721 caagtgcccc gatgttactg atggaaaaca tcgtgtggcg cgccacgata cttgtgttaa
      781 ttaaaccaaa ccagtctgtg ccaagtggat tgcttatgca tcgagtggcg agcccactga
      841 cctctgggcc ccttttttga actataaaaa gcggccaagt ctgatttcgg tctcacaatc
      901 atcaacatcg acttctcttt tcgcctcgat caacctaaac aacggtcact cagccatgaa
      961 ctggacaatg gaatatttcg ccttgccttg gatctttggc ctctagactc aatcaacgtc
     1021 aggataaagg tgagcatagg ttgctcactc actgtagcct ttttttccct tcttcttggt
     1081 ccagatggtt cccaaggctc tataaagtgt atctgtcagt tccacttcct tactccgaag
     1141 cgattcagag ttgtaaagtg cgtctgaccc cctgattata tctcaaagtc ccttcacaaa
     1201 ttcagacatg atgcgataaa gtgccggatt gtcattcgcg tcttgtgact gtatagaaag
     1261 tgccggcatc ctcttttctt ttcctctttg gccgagcgac tcagagttgt gaagcctgct
     1321 ggtatctgtc tgttcccttg ccctgataaa gaatatcgaa gagcatttct ttgattcaca
     1381 atacgttgac gaatggtgtg atggatccta agcagcgcca gccctttgaa cttgtgtcga
     1441 aaaaaaaaaa aaaacagcat atgatacgta aagggaagaa aaggatggag ctgactgttt
     1501 tgacaggcat aattttgagc gtgcttcatt ggccggttta tctgtattaa cacggaagag
     1561 atgtagaaac tagcttcctg gtttcagaga caaggagcca tgaagatcct gaggattcaa
     1621 cggcgttgct gagggtggga aggatggtat tgttgtctgt agatagatta atgggcatat
     1681 aatgtcaaaa aagatgattg acacgatgtc tgtctcgaat tcgcagtgtg ctaaaattgt
     1741 atcacttctc taatatgtat atatacaccc tctaaggaaa attaacccct taccgccttc
     1801 acgaattctt aaccctccca gacgtaaccg gaggggagga gctctcggat accgacggcg
     1861 gtgggctgac cgtcggagtc cttgacgatg gccttgatac cggggtggag gtcgaggagg
     1921 acctgtcgac atcgaccaca gggggagagg atacctcggt tctcgttacc gatggcgacg
     1981 atacaggtga ggttaccggc ggcggcggcg gcggcggtac cgaggacgac gagctcggca
     2041 cagggaccac cggtgaagtg gtagacgttg acaccctgtc gggagatgtt agtggagtaa
     2101 catgttgcgg gaaacggggt tacatacggt gaagattcga ccgtcggagg agagggcggc
     2161 ggaggcgacg gagtagtcct cggagatggg gatggagttg atggtggcgg tggctcgctc
     2221 gatgagggtg gactcctcct gggagagggg cttggccatg gcggccgcca tagacatgtt
     2281 gggcgagttt actaatggaa aaagacaatt agcaaagagg ctgcatatta ggagacgcgg
     2341 tgaagattgg ggaggacaat agggtccgtg caccaaaaag tcctgacggc agctctgttc
     2401 aacgcacatc catctcttcc agctatgcgt gtggcaccag cacggatgga ttgttgcacc
     2461 aaaaggacaa ggcgagatgg atgcaggtcg aggcgatgcg gtgctatcgg ccctcgttgt
     2521 cgaacgccac gtcgtcctct cggcgaccgt caatcctgga aggaaatgac caagcgtgca
     2581 cccagcatcc agcatccaac atccagcacc cagcacctag cacccatgat ccaatggccg
     2641 gcatgcagat cagcgtgggg agagcaccat ctagcagccc ctggcaggcc cagcattgtg
     2701 ctcggatgtg cgataggcgg tgggtgcgat aaggtggatt agtggaggat ggaggactga
     2761 gtggcgggca agggaagggg gcgggaagat gactcacctt gggcagtggg tgggggatgc
     2821 ggtatagggg atgtatgtgg atgctggcgg aggatagaag ctgtaagctg agctggtgcc
     2881 aacggtgacg ctgtgagagt ggtctcggcg accctgacgg cgactcggcg ttaccccgtg
     2941 tggcacaccg gacgcgcgtc gagtgacgtt gctgctattg ttgttaccat catcctctcc
     3001 tccgacaacc atacactcag tgcacaccat gccactcgaa tcctgcatgc ttatgtgagt
     3061 cctccccttc ccgtgcgccg ctctccagct cacatcctcg cagccatgga ctcaacccta
     3121 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3181 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     3241 tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     3301 tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag
     3361 ggttttccca gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac
     3421 tatagggcga attgggtacc gggccccccc tcgaggtcga cggtatcgat aagcttgata
     3481 tcgaattcct gcagcccggg ggatccacta gttctagagc ggccgccacc gcggtggagc
     3541 tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag
     3601 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     3661 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     3721 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     3781 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     3841 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     3901 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     3961 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4021 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4081 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4141 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4201 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4261 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4321 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4381 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     4441 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4501 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4561 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     4621 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     4681 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4741 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     4801 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     4861 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     4921 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     4981 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5041 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5101 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5161 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5221 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5281 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5341 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     5401 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     5461 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     5521 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     5581 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     5641 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     5701 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgc gccctgtagc
     5761 ggca
//
