LOCUS       sequence-461779-        8330 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Carries AID* tag, empty sgRNA, and HYGR marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8330)
  TITLE     C. neoformans AID2 degron system plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8330)
  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            "Unpublished"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: pBHM2650
FEATURES             Location/Qualifiers
     source          1..8330
                     /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(2771..3787)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="PELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYVL
                     RVNSCANGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPETE
                     LPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTVM
                     DDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGDS
                     QYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLGQLYQSLVDGNFD
                     DAAWAQGRCDAIVRSGAGTVGRTQIAHRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     primer_bind     complement(4605..4624)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4606..4624)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4631..4648)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4631..4647)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4640..4662)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4788..5243
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4875..4894)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5085..5106
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5374..6177)
                     /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(6178..6393)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6417..6436)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6536..6558
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6596..6614)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    6655..7158
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        7195..7299
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7300..7368,7369..8160)
                     /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(7518..7537)
                     /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 tcacgagttc ctattccttt gccctcggac gagtgctggg gcgtcggttt ccactatcgg
     2821 cgagtacttc tacacagcca tcggtccaga cggccgcgct tctgtgggcg atttgtgtac
     2881 gcccgacagt cccggctccg gatcggacga ttgcgtcgca tcgaccctgc gcccaagctg
     2941 catcatcgaa attgccgtca accaagctct gatagagttg gccaagacca atgcggagca
     3001 tatacgcccg gagccgcggc gatcctgcaa gctccggatg cctccgctcg aagtagcgcg
     3061 tctgctgctc catacaagcc aaccacggcc tccagaagaa gatgttggcg acctcgtatt
     3121 gggaatcccc gaacatcgcc tcgctccagt caatgaccgc tgttatgcgg ccattgtccg
     3181 tcaggacatt gttggagccg aaatccgcgt gcacgaggtg ccggacttcg gggcagtcct
     3241 cggcccaaag catcagctca tcgagagcct gcgcgacgga cgcactgacg gtgtcgtcca
     3301 tcacagtttg ccagtgatac acatggggat cagcaatcgc gcatatgaaa tcacgccatg
     3361 tagtgtattg accgattcct tgcggtccga atgggccgaa cccgctcgtc tggctaagat
     3421 cggccgcagc gatcgcatcc atggcctccg cgaccggctg cagaacagcg ggcagttcgg
     3481 tttcaggcag gtcttgcaac gtgacaccct gtgcacggcg ggagatgcaa taggtcaggc
     3541 tctcgctgaa ttccccaatg tcaagcactt ccggaatcgg gagcgcggcc gatgcaaagt
     3601 gccgataaac ataacgatct ttgtagaaac cattggcgca gctatttacc cgcaggacat
     3661 atccacgccc tcctacatcg aagctgaaag cacgagattc ttcgccctcc gagagctgca
     3721 tcaggtcgga gacgctgtcg aacttttcga tcagaaactt ctcgacagac gtcgcggtga
     3781 gttcaggtgg atcaccttct tcttccatag acatgttggg cgagtttact aatggaaaaa
     3841 gacaattagc aaagaggctg catattagga gacgcggtga agattgggga ggacaatagg
     3901 gtccgtgcac caaaaagtcc tgacggcagc tctgttcaac gcacatccat ctcttccagc
     3961 tatgcgtgtg gcaccagcac ggatggattg ttgcaccaaa aggacaaggc gagatggatg
     4021 caggtcgagg cgatgcggtg ctatcggccc tcgttgtcga acgccacgtc gtcctctcgg
     4081 cgaccgtcaa tcctggaagg aaatgaccaa gcgtgcaccc agcatccagc atccaacatc
     4141 cagcacccag cacctagcac ccatgatcca atggccggca tgcagatcag cgtggggaga
     4201 gcaccatcta gcagcccctg gcaggcccag cattgtgctc ggatgtgcga taggcggtgg
     4261 gtgcgataag gtggattagt ggaggatgga ggactgagtg gcgggcaagg gaagggggcg
     4321 ggaagatgac tcaccttggg cagtgggtgg gggatgcggt ataggggatg tatgtggatg
     4381 ctggcggagg atagaagctg taagctgagc tggtgccaac ggtgacgctg tgagagtggt
     4441 ctcggcgacc ctgacggcga ctcggcgtta ccccgtgtgg cacaccggac gcgcgtcgag
     4501 tgacgttgct gctattgttg ttaccatcat cctctcctcc gacaaccata cactcagtgc
     4561 acaccatgcc actcgaatcc tgcatgcggt ggagctccaa ttcgccctat agtgagtcgt
     4621 attacaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     4681 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     4741 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggacg cgccctgtag
     4801 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4861 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     4921 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4981 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     5041 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     5101 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     5161 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     5221 caaaatatta acgcttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt
     5281 atttcacacc gcatagggta ataactgata taattaaatt gaagctctaa tttgtgagtt
     5341 tagtatacat gcatttactt ataatacagt tttttagttt tgctggccgc atcttctcaa
     5401 atatgcttcc cagcctgctt ttctgtaacg ttcaccctct accttagcat cccttccctt
     5461 tgcaaatagt cctcttccaa caataataat gtcagatcct gtagagacca catcatccac
     5521 ggttctatac tgttgaccca atgcgtctcc cttgtcatct aaacccacac cgggtgtcat
     5581 aatcaaccaa tcgtaacctt catctcttcc acccatgtct ctttgagcaa taaagccgat
     5641 aacaaaatct ttgtcgctct tcgcaatgtc aacagtaccc ttagtatatt ctccagtaga
     5701 tagggagccc ttgcatgaca attctgctaa catcaaaagg cctctaggtt cctttgttac
     5761 ttcttctgcc gcctgcttca aaccgctaac aatacctggg cccaccacac cgtgtgcatt
     5821 cgtaatgtct gcccattctg ctattctgta tacacccgca gagtactgca atttgactgt
     5881 attaccaatg tcagcaaatt ttctgtcttc gaagagtaaa aaattgtact tggcggataa
     5941 tgcctttagc ggcttaactg tgccctccat ggaaaaatca gtcaagatat ccacatgtgt
     6001 ttttagtaaa caaattttgg gacctaatgc ttcaactaac tccagtaatt ccttggtggt
     6061 acgaacatcc aatgaagcac acaagtttgt ttgcttttcg tgcatgatat taaatagctt
     6121 ggcagcaaca ggactaggat gagtagcagc acgttcctta tatgtagctt tcgacatgat
     6181 ttatcttcgt ttcctgcagg tttttgttct gtgcagttgg gttaagaata ctgggcaatt
     6241 tcatgtttct tcaacactac atatgcgtat atataccaat ctaagtctgt gctccttcct
     6301 tcgttcttcc ttctgttcgg agattaccga atcaaaaaaa tttcaaggaa accgaaatca
     6361 aaaaaaagaa taaaaaaaaa atgatgaatt gaaaaggtgg tatggtgcac tctcagtaca
     6421 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     6481 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     6541 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     6601 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacggatcgc
     6661 ttgcctgtaa cttacacgcg cctcgtatct tttaatgatg gaataatttg ggaatttact
     6721 ctgtgtttat ttatttttat gttttgtatt tggattttag aaagtaaata aagaaggtag
     6781 aagagttacg gaatgaagaa aaaaaaataa acaaaggttt aaaaaatttc aacaaaaagc
     6841 gtactttaca tatatattta ttagacaaga aaagcagatt aaatagatat acattcgatt
     6901 aacgataagt aaaatgtaaa atcacaggat tttcgtgtgt ggtcttctac acagacaaga
     6961 tgaaacaatt cggcattaat acctgagagc aggaagagca agataaaagg tagtatttgt
     7021 tggcgatccc cctagagtct tttacatctt cggaaaacaa aaactatttt ttctttaatt
     7081 tcttttttta ctttctattt ttaatttata tatttatatt aaaaaattta aattataatt
     7141 atttttatag cacgtgatga aaaggaccca ggtggcactt ttcggggaaa tgtgcgcgga
     7201 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     7261 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     7321 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     7381 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     7441 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     7501 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     7561 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     7621 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     7681 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     7741 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     7801 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     7861 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     7921 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     7981 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     8041 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     8101 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     8161 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     8221 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     8281 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
//
