LOCUS       sequence-461132-        8082 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Carries AID* tag, empty sgRNA, and NEOR marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8082)
  TITLE     C. neoformans AID2 degron system plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8082)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pBHM2651
FEATURES             Location/Qualifiers
     source          1..8082
                     /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"
     CDS             1085..1216
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Morawska and Ulrich, 
                     2013)"
                     /label=AID*
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="PKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEAAAFVK"
     CDS             1229..1276
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     misc_RNA        complement(2167..2242)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(2766..3566)
                     /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(2877..2896)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3487..3506
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4357..4376)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4358..4376)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4383..4400)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4383..4399)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4392..4414)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4540..4995
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4627..4646)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4837..4858
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5126..5929)
                     /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(5930..6145)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6169..6188)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6288..6310
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6348..6366)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    6407..6910
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        6947..7051
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7052..7120,7121..7912)
                     /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(7270..7289)
                     /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 aggggttctg gatccggagg
     1081 tagtcctaaa gatccagcca aacctccggc caaggcacaa gttgtgggat ggccaccggt
     1141 gagatcatac cggaagaacg tgatggtttc ctgccaaaaa tcaagcggtg gcccggaggc
     1201 ggcggcgttc gtgaaggggt cagggtcaga ttataaagat gacgatgaca aggattataa
     1261 agatgacgat gacaagtaat tacttatgta gtgacccttc ttacgcgtaa tatgcatcgg
     1321 atgactgttg ctacttgtgc ttcttacgtt ttgtgtctta tttgctgatg tatacctctg
     1381 cgcttcaaaa tcttcattgc cgtgatatat cattccatgt cgtttgtccg ttattggatt
     1441 cccgatagtt gtccgcgaaa tcgaatattt ctcggctgac gacgtctctt gcaacaataa
     1501 aatggccatc actgttgaaa tttgcacgag agctataact tggaataacc ttcgttgctg
     1561 ctttatttta gtgctttcaa ctatttactc attctcaggt cgccaccatc gcattgatat
     1621 cgttccttcc atccttcctt tcctttagtc ggtcacgtct tccttaatcc gtcactcctt
     1681 ttttcagtcg ttcctggaga tcaccacgaa ataatctcga catgcactga ccatctcggt
     1741 tctgtggact gtcatgctaa gagagttcat gttgtaataa tagtttgttg catggaaaga
     1801 acggggaaag aagcaagaaa cagatgtaca gaattaccga tgtctctacg catccctgac
     1861 ctcgtccttc ctaccgcaac cgcacttcca cctgctcaca cgtcctcgtt aatgccttga
     1921 atctcagcca cacacttacc tcgcctcgac gcttgtcggg ccagggcacc cccttcatca
     1981 acctgggaag agttggcggg gcaaacggag gagggatggg gcaggaagag ggggatggcg
     2041 cacgtcgagt ggatttggaa gaaaaggggt tggtcgaggt ggaagtcgaa gcgaagatgt
     2101 tgaatgtcat gtttgggaac gggggatctt tagaagacta tgagattgaa cgagcttgat
     2161 aaaaaagcac cgactcggtg ccactttttc aagttgataa cggactagcc ttattttaac
     2221 ttgctatttc tagctctaaa acctaggcat gaggatatct cgcatctagc aacagtatac
     2281 cctgccggtg tatgtgcaat ttccactttg gcggataacg gaagcaaata agacggcggt
     2341 ccacgccgtc tcagtgtttt tttatttttt tcccgtcatc cattgttttt ctcctcacct
     2401 cttcattctc aacaaatggt tataattatc ggtcacacct gcaggttacc attgtccttg
     2461 cacgtatact ttgcatgcat gccgatcaat ttaaaaaata aatgaactgt aataatcatg
     2521 ctttgttttt agttctaatg caagggggat ccactagttg ccatgaagat cctgaggatt
     2581 caacggcgtt gctgagggtg ggaaggatgg tattgttgtc tgtagataga ttaatgggca
     2641 tataatgtca aaaaagatga ttgacacgat gtctgtctcg aattcgcagt gtgctaaaat
     2701 tgtatcactt ctctaatatg tatatataca ccctctaagg aaaattaacc ccttaccgcc
     2761 ttcactcaga agaactcgtc aagaaggcgg tagaaggcga tgcgctgcga atcgggagcg
     2821 gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata
     2881 tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg
     2941 atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg
     3001 gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct
     3061 ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc
     3121 cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga
     3181 tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca
     3241 aggtgggatg acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc
     3301 gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat
     3361 agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa
     3421 agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc
     3481 tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc
     3541 aatccatctt gttcaatcat agacatgttg ggcgagttta ctaatggaaa aagacaatta
     3601 gcaaagaggc tgcatattag gagacgcggt gaagattggg gaggacaata gggtccgtgc
     3661 accaaaaagt cctgacggca gctctgttca acgcacatcc atctcttcca gctatgcgtg
     3721 tggcaccagc acggatggat tgttgcacca aaaggacaag gcgagatgga tgcaggtcga
     3781 ggcgatgcgg tgctatcggc cctcgttgtc gaacgccacg ccgtcctctc ggcgaccgtc
     3841 aatcctggaa ggaagtgacc aagcgtgcac ccagcatcca gcatccaaca tccagcaccc
     3901 agcacctagc acccatgatc caatggccgg catgcagatc agcgtgggga gagcaccatc
     3961 tagcagcccc tggcaggccc agcattgtgc tcggatgtgc gataggcggt gggtgcgata
     4021 aggtggatta gtggaggatg gaggactgag tggcgggcaa gggaaggggg cgggaagatg
     4081 actcaccttg ggcagtgggt gggggatgcg gtatagggga tgtatgtgga tgctggcgga
     4141 ggatagaagc tgtaagctga gctggtgcca acggtgacgc tgtgagagtg gtctcggcga
     4201 ccctgacggc gactcggcgt taccccgtgt ggcacaccgg acgcgcgtcg agtgacgttg
     4261 ctgctattgt tgttaccatc atcctctcct ccgacaacca tacactcagt gcacaccatg
     4321 ccactcgaat cctgcatgcg gtggagctcc aattcgccct atagtgagtc gtattacaat
     4381 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     4441 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     4501 cgcccttccc aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat
     4561 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     4621 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     4681 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     4741 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     4801 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     4861 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     4921 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     4981 taacgcttac aatttcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     5041 ccgcataggg taataactga tataattaaa ttgaagctct aatttgtgag tttagtatac
     5101 atgcatttac ttataataca gttttttagt tttgctggcc gcatcttctc aaatatgctt
     5161 cccagcctgc ttttctgtaa cgttcaccct ctaccttagc atcccttccc tttgcaaata
     5221 gtcctcttcc aacaataata atgtcagatc ctgtagagac cacatcatcc acggttctat
     5281 actgttgacc caatgcgtct cccttgtcat ctaaacccac accgggtgtc ataatcaacc
     5341 aatcgtaacc ttcatctctt ccacccatgt ctctttgagc aataaagccg ataacaaaat
     5401 ctttgtcgct cttcgcaatg tcaacagtac ccttagtata ttctccagta gatagggagc
     5461 ccttgcatga caattctgct aacatcaaaa ggcctctagg ttcctttgtt acttcttctg
     5521 ccgcctgctt caaaccgcta acaatacctg ggcccaccac accgtgtgca ttcgtaatgt
     5581 ctgcccattc tgctattctg tatacacccg cagagtactg caatttgact gtattaccaa
     5641 tgtcagcaaa ttttctgtct tcgaagagta aaaaattgta cttggcggat aatgccttta
     5701 gcggcttaac tgtgccctcc atggaaaaat cagtcaagat atccacatgt gtttttagta
     5761 aacaaatttt gggacctaat gcttcaacta actccagtaa ttccttggtg gtacgaacat
     5821 ccaatgaagc acacaagttt gtttgctttt cgtgcatgat attaaatagc ttggcagcaa
     5881 caggactagg atgagtagca gcacgttcct tatatgtagc tttcgacatg atttatcttc
     5941 gtttcctgca ggtttttgtt ctgtgcagtt gggttaagaa tactgggcaa tttcatgttt
     6001 cttcaacact acatatgcgt atatatacca atctaagtct gtgctccttc cttcgttctt
     6061 ccttctgttc ggagattacc gaatcaaaaa aatttcaagg aaaccgaaat caaaaaaaag
     6121 aataaaaaaa aaatgatgaa ttgaaaaggt ggtatggtgc actctcagta caatctgctc
     6181 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     6241 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     6301 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     6361 cctattttta taggttaatg tcatgataat aatggtttct tagacggatc gcttgcctgt
     6421 aacttacacg cgcctcgtat cttttaatga tggaataatt tgggaattta ctctgtgttt
     6481 atttattttt atgttttgta tttggatttt agaaagtaaa taaagaaggt agaagagtta
     6541 cggaatgaag aaaaaaaaat aaacaaaggt ttaaaaaatt tcaacaaaaa gcgtacttta
     6601 catatatatt tattagacaa gaaaagcaga ttaaatagat atacattcga ttaacgataa
     6661 gtaaaatgta aaatcacagg attttcgtgt gtggtcttct acacagacaa gatgaaacaa
     6721 ttcggcatta atacctgaga gcaggaagag caagataaaa ggtagtattt gttggcgatc
     6781 cccctagagt cttttacatc ttcggaaaac aaaaactatt ttttctttaa tttctttttt
     6841 tactttctat ttttaattta tatatttata ttaaaaaatt taaattataa ttatttttat
     6901 agcacgtgat gaaaaggacc caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     6961 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     7021 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     7081 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     7141 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     7201 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     7261 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     7321 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     7381 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     7441 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     7501 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     7561 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     7621 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     7681 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     7741 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     7801 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     7861 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     7921 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     7981 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     8041 ccactgagcg tcagaccccg tagaaaagat caaaggatct tc
//
