LOCUS       sequence-478426-        8238 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Linker-Cno_mNeonGreen-CBP-2xFLAG-CnU6-sgRNA-NEOR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8238)
  AUTHORS   Nalley MJ, Banerjee S, Huang MY, Madhani HD
  TITLE     Near 100% efficient homology-dependent genome engineering in the 
            human fungal pathogen Cryptococcus neoformans
  JOURNAL   bioRxiv 2025.02.14.638277
REFERENCE   2  (bases 1 to 8238)
  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: "bioRxiv 
            2025.02.14.638277"
COMMENT     Sequence Label: pBHM2661
FEATURES             Location/Qualifiers
     source          1..8238
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     1058..1074
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1059..1075
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1093..1116
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     regulatory      1381..1390
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1846..1923
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     CDS             1936..1983
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     misc_RNA        complement(2323..2398)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(2922..3722)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MSMIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQG
                     RPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEE
                     HQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQD
                     IALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3033..3052)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3643..3662
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4513..4532)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4514..4532)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4539..4556)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4539..4555)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4548..4570)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4696..5151
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4783..4802)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4993..5014
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5282..6085)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(6086..6301)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6325..6344)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6444..6466
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6504..6522)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    6563..7066
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        7103..7207
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7208..7276,7277..8068)
                     /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(7426..7445)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagctcg aaattaaccc
     1021 tcactaaagg gaacaaaagc tggtaccggg ccccccctcg aggtcgacgg tatcgataag
     1081 cttggtggcg gactcgaagt cttgttccaa ggtcctggat ctggtatggt ctccaagggt
     1141 gaggaggaca acatggcctc cctccccgcc acccacgagc tccacatctt cggttccatc
     1201 aacggtgtcg acttcgacat ggtcggtcag ggtaccggta accccaacga cggttacgag
     1261 gagctcaacc tcaagtccac caagggtgac ctccagttct ccccctggat cctcgtcccc
     1321 cacatcggtt acggtttcca ccagtacctc ccctaccccg acggtatgtc ccccttccag
     1381 gccgccatgg tcgacggttc cggttaccag gtccaccgaa ccatgcagtt cgaggacggt
     1441 gcctccctca ccgtcaacta ccgatacacc tacgagggtt cccacatcaa gggtgaggcc
     1501 caggtcaagg gtaccggttt ccccgccgac ggtcccgtca tgaccaactc cctcaccgcc
     1561 gccgactggt gtcgatccaa gaagacctac cccaacgaca agaccatcat ctccaccttc
     1621 aagtggtcct acaccaccgg taacggtaag cgataccgat ccaccgcccg aaccacctac
     1681 accttcgcca agcccatggc cgccaactac ctcaagaacc agcccatgta cgtcttccga
     1741 aagaccgagc tcaagcactc caagaccgag ctcaacttca aggagtggca gaaggccttc
     1801 accgacgtca tgggtatgga cgagctctac aaggggtcag ggtcaaagag aagatggaaa
     1861 aagaatttca tagccgtctc agcagccaac cgctttaaga aaatctcatc ctccggggca
     1921 cttgggtcag ggtcagatta taaagatgac gatgacaagg attataaaga tgacgatgac
     1981 aagtaatctg gacatgtcaa ttgtacactg ccttataatg ccccatgcac ttccagtatt
     2041 agtcatttga agcgttgaaa gatctcttgg cagtgtgatc atggaaacaa tcccaaacag
     2101 cagcaaactg aaaacgttct ggtatgcaat ccatcccatc gtacaagctc tccaaatacc
     2161 tcttccaatc caaaaacggt ttagatgaaa gtctcgtctg atgcacttgt tagtgacacc
     2221 ccagaatttg atgatcttcg actcacctcg agcggccgcc agtgtgatgg atatctgcag
     2281 aattcgccct tgaatagagt cacttttgct agggataaaa aagcaccgac tcggtgccac
     2341 tttttcaagt tgataacgga ctagccttat tttaacttgc tatttctagc tctaaaacct
     2401 aggcatgagg atatctcgca tctagcaaca gtataccctg ccggtgtatg tgcaatttcc
     2461 actttggcgg ataacggaag caaataagac ggcggtccac gccgtctcag tgttttttta
     2521 tttttttccc gtcatccatt gtttttctcc tcacctcttc attctcaaca aatggttata
     2581 attatcggtc acacctgcag gttaccattg tccttgcacg tatactttgc atgcatgccg
     2641 atcaatttaa aaaataaatg aactgtaata atcatgcttt gtttttagtt ctaatgcaag
     2701 ggggatccac tagttgccat gaagatcctg aggattcaac ggcgttgctg agggtgggaa
     2761 ggatggtatt gttgtctgta gatagattaa tgggcatata atgtcaaaaa agatgattga
     2821 cacgatgtct gtctcgaatt cgcagtgtgc taaaattgta tcacttctct aatatgtata
     2881 tatacaccct ctaaggaaaa ttaacccctt accgccttca ctcagaagaa ctcgtcaaga
     2941 aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag
     3001 cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc
     3061 tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt
     3121 tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg
     3181 ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg
     3241 tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga
     3301 tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt
     3361 gcatcagcca tgatggatac tttctcggca ggagcaaggt gggatgacag gagatcctgc
     3421 cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca
     3481 gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt
     3541 tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac
     3601 agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat
     3661 agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatagac
     3721 atgttgggcg agtttactaa tggaaaaaga caattagcaa agaggctgca tattaggaga
     3781 cgcggtgaag attggggagg acaatagggt ccgtgcacca aaaagtcctg acggcagctc
     3841 tgttcaacgc acatccatct cttccagcta tgcgtgtggc accagcacgg atggattgtt
     3901 gcaccaaaag gacaaggcga gatggatgca ggtcgaggcg atgcggtgct atcggccctc
     3961 gttgtcgaac gccacgtcgt cctctcggcg accgtcaatc ctggaaggaa atgaccaagc
     4021 gtgcacccag catccagcat ccaacatcca gcacccagca cctagcaccc atgatccaat
     4081 ggccggcatg cagatcagcg tggggagagc accatctagc agcccctggc aggcccagca
     4141 ttgtgctcgg atgtgcgata ggcggtgggt gcgataaggt ggattagtgg aggatggagg
     4201 actgagtggc gggcaaggga agggggcggg aagatgactc accttgggca gtgggtgggg
     4261 gatgcggtat aggggatgta tgtggatgct ggcggaggat agaagctgta agctgagctg
     4321 gtgccaacgg tgacgctgtg agagtggtct cggcgaccct gacggcgact cggcgttacc
     4381 ccgtgtggca caccggacgc gcgtcgagtg acgttgctgc tattgttgtt accatcatcc
     4441 tctcctccga caaccataca ctcagtgcac accatgccac tcgaatcctg catgcggtgg
     4501 agctccaatt cgccctatag tgagtcgtat tacaattcac tggccgtcgt tttacaacgt
     4561 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     4621 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     4681 ctgaatggcg aatggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     4741 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     4801 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4861 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     4921 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4981 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     5041 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5101 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt tcctgatgcg
     5161 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atagggtaat aactgatata
     5221 attaaattga agctctaatt tgtgagttta gtatacatgc atttacttat aatacagttt
     5281 tttagttttg ctggccgcat cttctcaaat atgcttccca gcctgctttt ctgtaacgtt
     5341 caccctctac cttagcatcc cttccctttg caaatagtcc tcttccaaca ataataatgt
     5401 cagatcctgt agagaccaca tcatccacgg ttctatactg ttgacccaat gcgtctccct
     5461 tgtcatctaa acccacaccg ggtgtcataa tcaaccaatc gtaaccttca tctcttccac
     5521 ccatgtctct ttgagcaata aagccgataa caaaatcttt gtcgctcttc gcaatgtcaa
     5581 cagtaccctt agtatattct ccagtagata gggagccctt gcatgacaat tctgctaaca
     5641 tcaaaaggcc tctaggttcc tttgttactt cttctgccgc ctgcttcaaa ccgctaacaa
     5701 tacctgggcc caccacaccg tgtgcattcg taatgtctgc ccattctgct attctgtata
     5761 cacccgcaga gtactgcaat ttgactgtat taccaatgtc agcaaatttt ctgtcttcga
     5821 agagtaaaaa attgtacttg gcggataatg cctttagcgg cttaactgtg ccctccatgg
     5881 aaaaatcagt caagatatcc acatgtgttt ttagtaaaca aattttggga cctaatgctt
     5941 caactaactc cagtaattcc ttggtggtac gaacatccaa tgaagcacac aagtttgttt
     6001 gcttttcgtg catgatatta aatagcttgg cagcaacagg actaggatga gtagcagcac
     6061 gttccttata tgtagctttc gacatgattt atcttcgttt cctgcaggtt tttgttctgt
     6121 gcagttgggt taagaatact gggcaatttc atgtttcttc aacactacat atgcgtatat
     6181 ataccaatct aagtctgtgc tccttccttc gttcttcctt ctgttcggag attaccgaat
     6241 caaaaaaatt tcaaggaaac cgaaatcaaa aaaaagaata aaaaaaaaat gatgaattga
     6301 aaaggtggta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     6361 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     6421 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     6481 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     6541 gataataatg gtttcttaga cggatcgctt gcctgtaact tacacgcgcc tcgtatcttt
     6601 taatgatgga ataatttggg aatttactct gtgtttattt atttttatgt tttgtatttg
     6661 gattttagaa agtaaataaa gaaggtagaa gagttacgga atgaagaaaa aaaaataaac
     6721 aaaggtttaa aaaatttcaa caaaaagcgt actttacata tatatttatt agacaagaaa
     6781 agcagattaa atagatatac attcgattaa cgataagtaa aatgtaaaat cacaggattt
     6841 tcgtgtgtgg tcttctacac agacaagatg aaacaattcg gcattaatac ctgagagcag
     6901 gaagagcaag ataaaaggta gtatttgttg gcgatccccc tagagtcttt tacatcttcg
     6961 gaaaacaaaa actatttttt ctttaatttc tttttttact ttctattttt aatttatata
     7021 tttatattaa aaaatttaaa ttataattat ttttatagca cgtgatgaaa aggacccagg
     7081 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     7141 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     7201 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     7261 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     7321 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     7381 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     7441 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     7501 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     7561 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     7621 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     7681 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     7741 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     7801 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     7861 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     7921 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     7981 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     8041 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     8101 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     8161 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     8221 aaagatcaaa ggatcttc
//
