LOCUS       sequence-466522-        9482 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PCR to add remove nucleotides within intron of ACT1-CUP1 insert then
            restriction digest and ligation of new insert to backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9482)
  TITLE     Hoskins lab plasmids 2025
REFERENCE   2  (bases 1 to 9482)
  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"
COMMENT     Sequence Label: ACT1-CUP1 9bp dist
FEATURES             Location/Qualifiers
     source          1..9482
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        26..669
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     562..585
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     terminator      1511..1781
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     primer_bind     complement(1861..1880)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2079..2180
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     promoter        2509..2914
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2915..4009
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(4507..4524)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4507..4523)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4516..4538)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4732..4751)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4851..4873
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4911..4929)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4997..5101
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5102..5170,5171..5962)
                     /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(5320..5339)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6133..6721
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6622..6641
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6875..6892
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7009..7030
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7045..7050,7051..7068,7069..7075)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7083..7099
                     /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     7088..7110
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7107..7123
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7107..7123
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      7475..8817
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    7807..7854
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
ORIGIN
        1 taagcttggc tgcaggtcga gtttatcatt atcaatactg ccatttcaaa gaatacgtaa
       61 ataattaata gtagtgattt tcctaacttt atttagtcaa aaaattagcc ttttaattct
      121 gctgtaaccc gtacatgccc aaaatagggg gcgggttaca cagaatatat aacatcgtag
      181 gtgtctgggt gaacagttta ttcctggcat ccactaaata taatggagcc cgctttttaa
      241 gctggcatcc agaaaaaaaa agaatcccag caccaaaata ttgttttctt caccaaccat
      301 cagttcatag gtccattctc ttagcgcaac tacagagaac aggggcacaa acaggcaaaa
      361 aacgggcaca acctcaatgg agtgatgcaa cctgcctgga gtaaatgatg acacaaggca
      421 attgacccac gcatgtatct atctcatttt cttacacctt ctattacctt ctgctctctc
      481 tgatttggaa aaagctgaaa aaaaaggttg aaaccagttc cctgaaatta ttcccctact
      541 tgactaataa gtatataaag acggtaggta ttgattgtaa ttctgtaaat ctatttctta
      601 aacttcttaa attctacttt tatagttagt ctttttttta gttttaaaac accaagaact
      661 tagtttcgac ggatccccgg cgagctcgcc ccttttagat ttttcacgct tactgctttt
      721 ttcttcccaa gatcgaaaat ttactgaatt aacaatggat tctggtatgt tctagcgctt
      781 gcaccatccc atttaactgt aagaagaatt gcacggtccc aattgctcga gagatttctc
      841 ttttaccttt ttttactatt tttcactctc ccataacctc ctatattgac tgatctgtaa
      901 taaccacgat attattggaa taaatagggg cttgaaattt ggaaaaaaaa aaaactgaaa
      961 tattttcgtg ataagtgata gtgatattct tcttttattt gctactgtgt ctcatgtact
     1021 aacatcgatt atatagaggt tggtaccggg ttcagcgaat taattaactt ccaaaatgaa
     1081 ggtcatgagt gccaatgcca atgtggtagc tgcaaaaata atgaacaatg ccaaaaatca
     1141 tgtagctgcc caacggggtg taacagcgac gacaaatgcc cctgcggtaa caagtctgaa
     1201 gaaaccaaga aatcatgctg ctctgggaaa taaaacgaat agtctttaat atattcattt
     1261 aactatttgc tgtttttaat ttttaaaagg agaaggaagt ttaatcgacg attctactca
     1321 gtttgagtac acttatgtat tttgtttaga tactttgtta atttataggt atgacctgca
     1381 gcccaagctg gtcgacctag ataagtaatg atccgggatc tcccatgtct ctactggtgg
     1441 tggtgcttct ttggaattat tggaaggtaa ggaattgcca ggtgttgctt tcttatccga
     1501 aaagaaataa attgaattga attgaaatcg atagatcaat ttttttcttt tctctttccc
     1561 catcctttac gctaaaataa tagtttattt tattttttga atatttttta tttatatacg
     1621 tatatataga ctattattta tcttttaatg attattaaga tttttattaa aaaaaaattc
     1681 gctcctcttt taatgccttt atgcagtttt tttttcccat tcgatatttc tatgttcggg
     1741 ttcagcgtat tttaagttta ataactcgaa aattctgcgt tcgttaaagc tccggatcga
     1801 tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg
     1861 cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg
     1921 cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct
     1981 ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct
     2041 gcttcctaat gcaggagtcg cataagggag agcgtcgaaa ttcggtcgaa aaaagaaaag
     2101 gagagggcca agagggaggg cattggtgac tattgagcac gtgagtatac gtgattaagc
     2161 acacaaaggc agcttggagt atgtctgtta ttaatttcac aggtagttct ggtccattgg
     2221 tgaaagtttg cggcttgcag agcacagagg ccgcagaatg tgctctagtc gaggagaact
     2281 tctagtatat ctacatacct aatattattg ccttattaaa aatggaatcc caacaattac
     2341 atcaaaatcc acattctctt caaaatcaat tgtcctgtac ttccttgttc atgtgtgttc
     2401 aaaaacgtta tatttatagg ataattatac tctatttctc aacaagtaat tggttgtttg
     2461 gccgagcggt ctaaggcgcc tgattcaaga aatatcttga ccgcagttaa ctgtgggaat
     2521 actcaggtat cgtaagatgc aagagttcga atctcttagc aaccattatt tttttcctca
     2581 acataacgag aacacacagg ggcgctatcg cacagaatca aattcgatga ctggaaattt
     2641 tttgttaatt tcagaggtcg cctgacgcat ataccttttt caactgaaaa attgggagaa
     2701 aaaggaaagg tgagagcgcc ggaaccggct tttcatatag aatagagaag cgttcatgac
     2761 taaatgcttg catcacaata cttgaagttg acaatattat ttaaggacct attgtttttt
     2821 ccaataggtg gttagcaatc gtcttacttt ctaacttttc ttacctttta catttcagca
     2881 atatatatat atatttcaag gatataccat tctaatgtct gcccctaaga agatcgtcgt
     2941 tttgccaggt gaccacgttg gtcaagaaat cacagccgaa gccattaagg ttcttaaagc
     3001 tatttctgat gttcgttcca atgtcaagtt cgatttcgaa aatcatttaa ttggtggtgc
     3061 tgctatcgat gctacaggtg ttccacttcc agatgaggcg ctggaagcct ccaagaaggc
     3121 tgatgccgtt ttgttaggtg ctgtgggtgg tcctaaatgg ggtaccggta gtgttagacc
     3181 tgaacaaggt ttactaaaaa tccgtaaaga acttcaattg tacgccaact taagaccatg
     3241 taactttgca tccgactctc ttttagactt atctccaatc aagccacaat ttgctaaagg
     3301 tactgacttc gttgttgtca gagaattagt gggaggtatt tactttggta agagaaagga
     3361 agacgatggt gatggtgtcg cttgggatag tgaacaatac accgttccag aagtgcaaag
     3421 aatcacaaga atggccgctt tcatggccct acaacatgag ccaccattgc ctatttggtc
     3481 cttggataaa gctaatgttt tggcctcttc aagattatgg agaaaaactg tggaggaaac
     3541 catcaagaac gaattcccta cattgaaggt tcaacatcaa ttgattgatt ctgccgccat
     3601 gatcctagtt aagaacccaa cccacctaaa tggtattata atcaccagca acatgtttgg
     3661 tgatatcatc tccgatgaag cctccgttat cccaggttcc ttgggtttgt tgccatctgc
     3721 gtccttggcc tctttgccag acaagaacac cgcatttggt ttgtacgaac catgccacgg
     3781 ttctgctcca gatttgccaa agaataaggt caaccctatc gccactatct tgtctgctgc
     3841 aatgatgttg aaattgtcat tgaacttgcc tgaagaaggt aaggccattg aagatgcagt
     3901 taaaaaggtt ttggatgcag gtatcagaac tggtgattta ggtggttcca acagtaccac
     3961 cgaagtcggt gatgctgtcg ccgaagaagt taagaaaatc cttgcttaaa aagattctct
     4021 ttttttatga tatttgtaca taaactttat aaatgaaatt cataatagaa acgacacgaa
     4081 attacaaaat ggaatatgtt catagggtag acgaaactat atacgcaatc tacatacatt
     4141 tatcaagaag gagaaaaagg aggatgtaaa ggaatacagg taagcaaatt gatactaatg
     4201 gctcaacgtg ataaggaaaa agaattgcac tttaacatta atattgacaa ggaggagggc
     4261 accacacaaa aagttaggtg taacagaaaa tcatgaaact atgattccta atttatatat
     4321 tggaggattt tctctaaaaa aaaaaaaata caacaaataa aaaacactca atgacctgac
     4381 catttgatgg agtttaagtc aataccttct tgaaccattt cccataatgg tgaaagttcc
     4441 ctcaagaatt ttactctgtc agaaacggcc ttaacgacgt agtcgagatc tgcatgcaag
     4501 cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     4561 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     4621 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
     4681 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     4741 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     4801 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     4861 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     4921 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     4981 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5041 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5101 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5161 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5221 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5281 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5341 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5401 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5461 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5521 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     5581 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     5641 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     5701 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     5761 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     5821 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     5881 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     5941 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6001 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6061 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6121 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6181 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6241 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6301 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6361 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6421 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6481 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6541 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     6601 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     6661 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     6721 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     6781 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     6841 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     6901 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     6961 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     7021 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     7081 aattgtgagc ggataacaat ttcacacagg aaacagctat gacatgatta cgaattctga
     7141 accagtccta aaacgagtaa ataggaccgg caattcttca agcaataaac aggaatacca
     7201 attattaaaa gataacttag tcagatcgta caataaagct ttgaagaaaa atgcgcctta
     7261 ttcaatcttt gctataaaaa atggcccaaa atctcacatt ggaagacatt tgatgacctc
     7321 atttctttca atgaagggcc taacggagtt gactaatgtt gtgggaaatt ggagcgataa
     7381 gcgtgcttct gccgtggcca ggacaacgta tactcatcag ataacagcaa tacctgatca
     7441 ctacttcgca ctagtttctc ggtactatgc atatgatcca atatcaaagg aaatgatagc
     7501 attgaaggat gagactaatc caattgagga gtggcagcat atagaacagc taaagggtag
     7561 tgctgaagga agcatacgat accccgcatg gaatgggata atatcacagg aggtactaga
     7621 ctacctttca tcctacataa atagacgcat ataagtacgc atttaagcat aaacacgcac
     7681 tatgccgttc ttctcatgta tatatatata caggcaacac gcagatatag gtgcgacgtg
     7741 aacagtgagc tgtatgtgcg cagctcgcgt tgcattttcg gaagcgctcg ttttcggaaa
     7801 cgctttgaag ttcctattcc gaagttccta ttctctagaa agtataggaa cttcagagcg
     7861 cttttgaaaa ccaaaagcgc tctgaagacg cactttcaaa aaaccaaaaa cgcaccggac
     7921 tgtaacgagc tactaaaata ttgcgaatac cgcttccaca aacattgctc aaaagtatct
     7981 ctttgctata tatctctgtg ctatatccct atataaccta cccatccacc tttcgctcct
     8041 tgaacttgca tctaaactcg acctctacat tttttatgtt tatctctagt attactcttt
     8101 agacaaaaaa attgtagtaa gaactattca tagagtgaat cgaaaacaat acgaaaatgt
     8161 aaacatttcc tatacgtagt atatagagac aaaatagaag aaaccgttca taattttctg
     8221 accaatgaag aatcatcaac gctatcactt tctgttcaca aagtatgcgc aatccacatc
     8281 ggtatagaat ataatcgggg atgcctttat cttgaaaaaa tgcacccgca gcttcgctag
     8341 taatcagtaa acgcgggaag tggagtcagg ctttttttat ggaagagaaa atagacacca
     8401 aagtagcctt cttctaacct taacggacct acagtgcaaa aagttatcaa gagactgcat
     8461 tatagagcgc acaaaggaga aaaaaagtaa tctaagatgc tttgttagaa aaatagcgct
     8521 ctcgggatgc atttttgtag aacaaaaaag aagtatagat tctttgttgg taaaatagcg
     8581 ctctcgcgtt gcatttctgt tctgtaaaaa tgcagctcag attctttgtt tgaaaaatta
     8641 gcgctctcgc gttgcatttt tgttttacaa aaatgaagca cagattcttc gttggtaaaa
     8701 tagcgctttc gcgttgcatt tctgttctgt aaaaatgcag ctcagattct ttgtttgaaa
     8761 aattagcgct ctcgcgttgc atttttgttc tacaaaatga agcacagatg cttcgttaac
     8821 aaagatatgc tattgaagtg caagatggaa acgcagaaaa tgaaccgggg atgcgacgtg
     8881 caagattacc tatgcaatag atgcaatagt ttctccagga accgaaatac atacattgtc
     8941 ttccgtaaag cgctagacta tatattatta tacaggttca aatatactat ctgtttcagg
     9001 gaaaactccc aggttcggat gttcaaaatt caatgatggg taacaagtac gatcgtaaat
     9061 ctgtaaaaca gtttgtcgga tattaggctg tatctcctca aagcgtattc gaatatcatt
     9121 gagaagctgc agctattcat gcaactgcta aatggtctac tgctcaatta aagaaaacac
     9181 aggcagagca ggctgcccgg gcaaaagctg cagcgtcaca tcggataata atgatggcag
     9241 ccattgtaga agtgcctttt gcatttctag tctctttctc ggtctagcta gttttactac
     9301 atcgcgaaga tagaatctta gatcacactg cctttgctga gctggatcaa tagagtaaca
     9361 aaagagtggt aaggcctcgt taaaggacaa ggacctgagc ggaagtgtat cgtacagtag
     9421 acggagtata ctagtatagt ctatagtccg tggaattctc atgtttgaca gcttatcatc
     9481 ga
//
