LOCUS       sequence-459927-        7869 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Carries AID* tag, empty sgRNA, and NATR marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7869)
  TITLE     C. neoformans AID2 degron system plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7869)
  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            "Unpublished"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: pBHM2644
FEATURES             Location/Qualifiers
     source          1..7869
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    69..572
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        609..713
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(714..782,783..1574)
                     /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(932..951)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1745..2333
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2234..2253
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2487..2504
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2621..2642
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2657..2662,2663..2680,2681..2687)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2695..2711
                     /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     2700..2722
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2719..2735
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2719..2735
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2756..2774
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             2829..2960
                     /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             2973..3020
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     misc_RNA        complement(3911..3986)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(4516..5079)
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="TLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFTLR
                     EVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRRLT
                     VEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTLCG
                     LDTALYDGTASDGEQALYMSMPCP"
     primer_bind     complement(5888..5907)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5889..5907)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5914..5931)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5914..5930)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5923..5945)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6071..6526
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6158..6177)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6368..6389
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6657..7460)
                     /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(7461..7676)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7700..7719)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7819..7841
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat gatggaataa
      121 tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt ttagaaagta
      181 aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag gtttaaaaaa
      241 tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca gattaaatag
      301 atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt gtgtggtctt
      361 ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag agcaagataa
      421 aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa acaaaaacta
      481 ttttttcttt aatttctttt tttactttct atttttaatt tatatattta tattaaaaaa
      541 tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc acttttcggg
      601 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
      661 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
      721 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
      781 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
      841 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
      901 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
      961 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1021 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1081 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1141 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1201 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1261 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1321 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     1381 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     1441 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     1501 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     1561 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     1621 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     1681 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     1741 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     1801 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     1861 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     1921 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     1981 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     2041 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     2101 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     2161 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     2221 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     2281 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     2341 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     2401 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     2461 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     2521 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     2581 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     2641 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     2701 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag ctcgaaatta
     2761 accctcacta aagggaacaa aagctggtac cgggcccccc ctcgaggggt tctggatccg
     2821 gaggtagtcc taaagatcca gccaaacctc cggccaaggc acaagttgtg ggatggccac
     2881 cggtgagatc ataccggaag aacgtgatgg tttcctgcca aaaatcaagc ggtggcccgg
     2941 aggcggcggc gttcgtgaag gggtcagggt cagattataa agatgacgat gacaaggatt
     3001 ataaagatga cgatgacaag taattactta tgtagtgacc cttcttacgc gtaatatgca
     3061 tcggatgact gttgctactt gtgcttctta cgttttgtgt cttatttgct gatgtatacc
     3121 tctgcgcttc aaaatcttca ttgccgtgat atatcattcc atgtcgtttg tccgttattg
     3181 gattcccgat agttgtccgc gaaatcgaat atttctcggc tgacgacgtc tcttgcaaca
     3241 ataaaatggc catcactgtt gaaatttgca cgagagctat aacttggaat aaccttcgtt
     3301 gctgctttat tttagtgctt tcaactattt actcattctc aggtcgccac catcgcattg
     3361 atatcgttcc ttccatcctt cctttccttt agtcggtcac gtcttcctta atccgtcact
     3421 ccttttttca gtcgttcctg gagatcacca cgaaataatc tcgacatgca ctgaccatct
     3481 cggttctgtg gactgtcatg ctaagagagt tcatgttgta ataatagttt gttgcatgga
     3541 aagaacgggg aaagaagcaa gaaacagatg tacagaatta ccgatgtctc tacgcatccc
     3601 tgacctcgtc cttcctaccg caaccgcact tccacctgct cacacgtcct cgttaatgcc
     3661 ttgaatctca gccacacact tacctcgcct cgacgcttgt cgggccaggg cacccccttc
     3721 atcaacctgg gaagagttgg cggggcaaac ggaggaggga tggggcagga agagggggat
     3781 ggcgcacgtc gagtggattt ggaagaaaag gggttggtcg aggtggaagt cgaagcgaag
     3841 atgttgaatg tcatgtttgg gaacggggga tctttagaag actatgagat tgaacgagct
     3901 tgataaaaaa gcaccgactc ggtgccactt tttcaagttg ataacggact agccttattt
     3961 taacttgcta tttctagctc taaaacctag gcatgaggat atctcgcatc tagcaacagt
     4021 ataccctgcc ggtgtatgtg caatttccac tttggcggat aacggaagca aataagacgg
     4081 cggtccacgc cgtctcagtg tttttttatt tttttcccgt catccattgt ttttctcctc
     4141 acctcttcat tctcaacaaa tggttataat tatcggtcac acctgcaggt taccattgtc
     4201 cttgcacgta tactttgcat gcatgccgat caatttaaaa aataaatgaa ctgtaataat
     4261 catgctttgt ttttagttct aatgcaaggg ggatccacta gttgccatga agatcctgag
     4321 gattcaacgg cgttgctgag ggtgggaagg atggtattgt tgtctgtaga tagattaatg
     4381 ggcatataat gtcaaaaaag atgattgaca cgatgtctgt ctcgaattcg cagtgtgcta
     4441 aaattgtatc acttctctaa tatgtatata tacaccctct aaggaaaatt aaccccttac
     4501 cgccttcacg aattcttagg ggcagggcat gctcatgtag agcgcctgct cgccgtccga
     4561 ggcggtgccg tcgtacaggg cggtgtccag gccgcagagg gtgaacccca tccgccggta
     4621 cgcgtggata gccggtgcgt taacgttggt gacctccagc cagaggtgcc cggcgccccg
     4681 ctcgcgggcg aactccgtcg cgagccccat caacgcgcgc ccgaccccgt gcccccggtg
     4741 ctccggggcg acctcgatgt cctcgacggt cagccggcgg ttccagccgg agtacgagac
     4801 gaccacgaag cccgccaggt cgccgtcgtc cccgtacgcg acgaacgtcc gggagtccgg
     4861 gtcgccgtcc tccccgtcgt ccgattcgtc gtccgattcg tcgtcgggga acaccttggt
     4921 caggggcggg tccaccggca cctcccgcag ggtgaagccg tccccggtgg cggtgacgcg
     4981 gaagacggtg tcggtggtga aggacccatc cagtgcctcg atggcctcgg cgtcccccgg
     5041 gacactggtg cggtaccggt aagccgtgtc gtcaagagtg gcggccgcca tagacatgtt
     5101 gggcgagttt actaatggaa aaagacaatt agcaaagagg ctgcatatta ggagacgcgg
     5161 tgaagattgg ggaggacaat agggtccgtg caccaaaaag tcctgacggc agctctgttc
     5221 aacgcacatc catctcttcc agctatgcgt gtggcaccag cacggatgga ttgttgcacc
     5281 aaaaggacaa ggcgagatgg atgcaggtcg aggcgatgcg gtgctatcgg ccctcgttgt
     5341 cgaacgccac gtcgtcctct cggcgaccgt caatcctgga aggaaatgac caagcgtgca
     5401 cccagcatcc agcatccaac atccagcacc cagcacctag cacccatgat ccaatggccg
     5461 gcatgcagat cagcgtgggg agagcaccat ctagcagccc ctggcaggcc cagcattgtg
     5521 ctcggatgtg cgataggcgg tgggtgcgat aaggtggatt agtggaggat ggaggactga
     5581 gtggcgggca agggaagggg gcgggaagat gactcacctt gggcagtggg tgggggatgc
     5641 ggtatagggg atgtatgtgg atgctggcgg aggatagaag ctgtaagctg agctggtgcc
     5701 aacggtgacg ctgtgagagt ggtctcggcg accctgacgg cgactcggcg ttaccccgtg
     5761 tggcacaccg gacgcgcgtc gagtgacgtt gctgctattg ttgttaccat catcctctcc
     5821 tccgacaacc atacactcag tgcacaccat gccactcgaa tcctgcatgc ggtggagctc
     5881 caattcgccc tatagtgagt cgtattacaa ttcactggcc gtcgttttac aacgtcgtga
     5941 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     6001 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     6061 tggcgaatgg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     6121 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     6181 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     6241 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     6301 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     6361 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     6421 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     6481 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttcctg atgcggtatt
     6541 ttctccttac gcatctgtgc ggtatttcac accgcatagg gtaataactg atataattaa
     6601 attgaagctc taatttgtga gtttagtata catgcattta cttataatac agttttttag
     6661 ttttgctggc cgcatcttct caaatatgct tcccagcctg cttttctgta acgttcaccc
     6721 tctaccttag catcccttcc ctttgcaaat agtcctcttc caacaataat aatgtcagat
     6781 cctgtagaga ccacatcatc cacggttcta tactgttgac ccaatgcgtc tcccttgtca
     6841 tctaaaccca caccgggtgt cataatcaac caatcgtaac cttcatctct tccacccatg
     6901 tctctttgag caataaagcc gataacaaaa tctttgtcgc tcttcgcaat gtcaacagta
     6961 cccttagtat attctccagt agatagggag cccttgcatg acaattctgc taacatcaaa
     7021 aggcctctag gttcctttgt tacttcttct gccgcctgct tcaaaccgct aacaatacct
     7081 gggcccacca caccgtgtgc attcgtaatg tctgcccatt ctgctattct gtatacaccc
     7141 gcagagtact gcaatttgac tgtattacca atgtcagcaa attttctgtc ttcgaagagt
     7201 aaaaaattgt acttggcgga taatgccttt agcggcttaa ctgtgccctc catggaaaaa
     7261 tcagtcaaga tatccacatg tgtttttagt aaacaaattt tgggacctaa tgcttcaact
     7321 aactccagta attccttggt ggtacgaaca tccaatgaag cacacaagtt tgtttgcttt
     7381 tcgtgcatga tattaaatag cttggcagca acaggactag gatgagtagc agcacgttcc
     7441 ttatatgtag ctttcgacat gatttatctt cgtttcctgc aggtttttgt tctgtgcagt
     7501 tgggttaaga atactgggca atttcatgtt tcttcaacac tacatatgcg tatatatacc
     7561 aatctaagtc tgtgctcctt ccttcgttct tccttctgtt cggagattac cgaatcaaaa
     7621 aaatttcaag gaaaccgaaa tcaaaaaaaa gaataaaaaa aaaatgatga attgaaaagg
     7681 tggtatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     7741 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     7801 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     7861 aacgcgcga
//
