LOCUS       sequence-459934-        7941 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Carries mAID_opt tag, empty sgRNA, and NATR marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7941)
  TITLE     C. neoformans AID2 degron system plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7941)
  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            "Unpublished"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: pBHM2646
FEATURES             Location/Qualifiers
     source          1..7941
                     /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..3032
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             3045..3092
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     misc_RNA        complement(3983..4058)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(4588..5151)
                     /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(5960..5979)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5961..5979)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5986..6003)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5986..6002)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5995..6017)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6143..6598
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6230..6249)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6440..6461
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6729..7532)
                     /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(7533..7748)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7772..7791)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7891..7913
                     /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 gaggtagtaa ggagaagtcc gcctgtccca aggaccccgc caagcccccc gccaaggccc
     2881 aggtcgtcgg ttggcccccc gtccgatcct accgaaagaa cgtcatggtc tcctgtcaga
     2941 agtcctccgg tggtcccgag gccgccgcct tcgtcaaggt ctccatggac ggtgccccct
     3001 acctccgaaa gatcgacctc cgaatgtaca aggggtcagg gtcagattat aaagatgacg
     3061 atgacaagga ttataaagat gacgatgaca agtaattact tatgtagtga cccttcttac
     3121 gcgtaatatg catcggatga ctgttgctac ttgtgcttct tacgttttgt gtcttatttg
     3181 ctgatgtata cctctgcgct tcaaaatctt cattgccgtg atatatcatt ccatgtcgtt
     3241 tgtccgttat tggattcccg atagttgtcc gcgaaatcga atatttctcg gctgacgacg
     3301 tctcttgcaa caataaaatg gccatcactg ttgaaatttg cacgagagct ataacttgga
     3361 ataaccttcg ttgctgcttt attttagtgc tttcaactat ttactcattc tcaggtcgcc
     3421 accatcgcat tgatatcgtt ccttccatcc ttcctttcct ttagtcggtc acgtcttcct
     3481 taatccgtca ctcctttttt cagtcgttcc tggagatcac cacgaaataa tctcgacatg
     3541 cactgaccat ctcggttctg tggactgtca tgctaagaga gttcatgttg taataatagt
     3601 ttgttgcatg gaaagaacgg ggaaagaagc aagaaacaga tgtacagaat taccgatgtc
     3661 tctacgcatc cctgacctcg tccttcctac cgcaaccgca cttccacctg ctcacacgtc
     3721 ctcgttaatg ccttgaatct cagccacaca cttacctcgc ctcgacgctt gtcgggccag
     3781 ggcaccccct tcatcaacct gggaagagtt ggcggggcaa acggaggagg gatggggcag
     3841 gaagaggggg atggcgcacg tcgagtggat ttggaagaaa aggggttggt cgaggtggaa
     3901 gtcgaagcga agatgttgaa tgtcatgttt gggaacgggg gatctttaga agactatgag
     3961 attgaacgag cttgataaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga
     4021 ctagccttat tttaacttgc tatttctagc tctaaaacct aggcatgagg atatctcgca
     4081 tctagcaaca gtataccctg ccggtgtatg tgcaatttcc actttggcgg ataacggaag
     4141 caaataagac ggcggtccac gccgtctcag tgttttttta tttttttccc gtcatccatt
     4201 gtttttctcc tcacctcttc attctcaaca aatggttata attatcggtc acacctgcag
     4261 gttaccattg tccttgcacg tatactttgc atgcatgccg atcaatttaa aaaataaatg
     4321 aactgtaata atcatgcttt gtttttagtt ctaatgcaag ggggatccac tagttgccat
     4381 gaagatcctg aggattcaac ggcgttgctg agggtgggaa ggatggtatt gttgtctgta
     4441 gatagattaa tgggcatata atgtcaaaaa agatgattga cacgatgtct gtctcgaatt
     4501 cgcagtgtgc taaaattgta tcacttctct aatatgtata tatacaccct ctaaggaaaa
     4561 ttaacccctt accgccttca cgaattctta ggggcagggc atgctcatgt agagcgcctg
     4621 ctcgccgtcc gaggcggtgc cgtcgtacag ggcggtgtcc aggccgcaga gggtgaaccc
     4681 catccgccgg tacgcgtgga tagccggtgc gttaacgttg gtgacctcca gccagaggtg
     4741 cccggcgccc cgctcgcggg cgaactccgt cgcgagcccc atcaacgcgc gcccgacccc
     4801 gtgcccccgg tgctccgggg cgacctcgat gtcctcgacg gtcagccggc ggttccagcc
     4861 ggagtacgag acgaccacga agcccgccag gtcgccgtcg tccccgtacg cgacgaacgt
     4921 ccgggagtcc gggtcgccgt cctccccgtc gtccgattcg tcgtccgatt cgtcgtcggg
     4981 gaacaccttg gtcaggggcg ggtccaccgg cacctcccgc agggtgaagc cgtccccggt
     5041 ggcggtgacg cggaagacgg tgtcggtggt gaaggaccca tccagtgcct cgatggcctc
     5101 ggcgtccccc gggacactgg tgcggtaccg gtaagccgtg tcgtcaagag tggcggccgc
     5161 catagacatg ttgggcgagt ttactaatgg aaaaagacaa ttagcaaaga ggctgcatat
     5221 taggagacgc ggtgaagatt ggggaggaca atagggtccg tgcaccaaaa agtcctgacg
     5281 gcagctctgt tcaacgcaca tccatctctt ccagctatgc gtgtggcacc agcacggatg
     5341 gattgttgca ccaaaaggac aaggcgagat ggatgcaggt cgaggcgatg cggtgctatc
     5401 ggccctcgtt gtcgaacgcc acgtcgtcct ctcggcgacc gtcaatcctg gaaggaaatg
     5461 accaagcgtg cacccagcat ccagcatcca acatccagca cccagcacct agcacccatg
     5521 atccaatggc cggcatgcag atcagcgtgg ggagagcacc atctagcagc ccctggcagg
     5581 cccagcattg tgctcggatg tgcgataggc ggtgggtgcg ataaggtgga ttagtggagg
     5641 atggaggact gagtggcggg caagggaagg gggcgggaag atgactcacc ttgggcagtg
     5701 ggtgggggat gcggtatagg ggatgtatgt ggatgctggc ggaggataga agctgtaagc
     5761 tgagctggtg ccaacggtga cgctgtgaga gtggtctcgg cgaccctgac ggcgactcgg
     5821 cgttaccccg tgtggcacac cggacgcgcg tcgagtgacg ttgctgctat tgttgttacc
     5881 atcatcctct cctccgacaa ccatacactc agtgcacacc atgccactcg aatcctgcat
     5941 gcggtggagc tccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt
     6001 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     6061 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     6121 gcgcagcctg aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     6181 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     6241 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     6301 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     6361 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     6421 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     6481 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     6541 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc
     6601 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata gggtaataac
     6661 tgatataatt aaattgaagc tctaatttgt gagtttagta tacatgcatt tacttataat
     6721 acagtttttt agttttgctg gccgcatctt ctcaaatatg cttcccagcc tgcttttctg
     6781 taacgttcac cctctacctt agcatccctt ccctttgcaa atagtcctct tccaacaata
     6841 ataatgtcag atcctgtaga gaccacatca tccacggttc tatactgttg acccaatgcg
     6901 tctcccttgt catctaaacc cacaccgggt gtcataatca accaatcgta accttcatct
     6961 cttccaccca tgtctctttg agcaataaag ccgataacaa aatctttgtc gctcttcgca
     7021 atgtcaacag tacccttagt atattctcca gtagataggg agcccttgca tgacaattct
     7081 gctaacatca aaaggcctct aggttccttt gttacttctt ctgccgcctg cttcaaaccg
     7141 ctaacaatac ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt
     7201 ctgtatacac ccgcagagta ctgcaatttg actgtattac caatgtcagc aaattttctg
     7261 tcttcgaaga gtaaaaaatt gtacttggcg gataatgcct ttagcggctt aactgtgccc
     7321 tccatggaaa aatcagtcaa gatatccaca tgtgttttta gtaaacaaat tttgggacct
     7381 aatgcttcaa ctaactccag taattccttg gtggtacgaa catccaatga agcacacaag
     7441 tttgtttgct tttcgtgcat gatattaaat agcttggcag caacaggact aggatgagta
     7501 gcagcacgtt ccttatatgt agctttcgac atgatttatc ttcgtttcct gcaggttttt
     7561 gttctgtgca gttgggttaa gaatactggg caatttcatg tttcttcaac actacatatg
     7621 cgtatatata ccaatctaag tctgtgctcc ttccttcgtt cttccttctg ttcggagatt
     7681 accgaatcaa aaaaatttca aggaaaccga aatcaaaaaa aagaataaaa aaaaaatgat
     7741 gaattgaaaa ggtggtatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     7801 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     7861 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     7921 cgtcatcacc gaaacgcgcg a
//
