LOCUS       sequence-466529-        9489 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 9489)
  TITLE     Hoskins lab plasmids 2025
REFERENCE   2  (bases 1 to 9489)
  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 15bp dist
FEATURES             Location/Qualifiers
     source          1..9489
                     /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      1518..1788
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     primer_bind     complement(1868..1887)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2086..2187
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     promoter        2516..2921
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2922..4016
                     /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(4514..4531)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4514..4530)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4523..4545)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4739..4758)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4858..4880
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4918..4936)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5004..5108
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5109..5177,5178..5969)
                     /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(5327..5346)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6140..6728
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6629..6648
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6882..6899
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7016..7037
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7052..7057,7058..7075,7076..7082)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7090..7106
                     /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     7095..7117
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7114..7130
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7114..7130
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      7482..8824
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    7814..7861
                     /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 aaaaactgaa
      961 atattttcgt gataagtgat agtgatattc ttcttttatt tgctactgtg tctcatgtac
     1021 taacatcgat tatatgttta tagaggttgg taccgggttc agcgaattaa ttaacttcca
     1081 aaatgaaggt catgagtgcc aatgccaatg tggtagctgc aaaaataatg aacaatgcca
     1141 aaaatcatgt agctgcccaa cggggtgtaa cagcgacgac aaatgcccct gcggtaacaa
     1201 gtctgaagaa accaagaaat catgctgctc tgggaaataa aacgaatagt ctttaatata
     1261 ttcatttaac tatttgctgt ttttaatttt taaaaggaga aggaagttta atcgacgatt
     1321 ctactcagtt tgagtacact tatgtatttt gtttagatac tttgttaatt tataggtatg
     1381 acctgcagcc caagctggtc gacctagata agtaatgatc cgggatctcc catgtctcta
     1441 ctggtggtgg tgcttctttg gaattattgg aaggtaagga attgccaggt gttgctttct
     1501 tatccgaaaa gaaataaatt gaattgaatt gaaatcgata gatcaatttt tttcttttct
     1561 ctttccccat cctttacgct aaaataatag tttattttat tttttgaata ttttttattt
     1621 atatacgtat atatagacta ttatttatct tttaatgatt attaagattt ttattaaaaa
     1681 aaaattcgct cctcttttaa tgcctttatg cagttttttt ttcccattcg atatttctat
     1741 gttcgggttc agcgtatttt aagtttaata actcgaaaat tctgcgttcg ttaaagctcc
     1801 ggatcgatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt
     1861 gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc
     1921 atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc
     1981 gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta
     2041 ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgaaattc ggtcgaaaaa
     2101 agaaaaggag agggccaaga gggagggcat tggtgactat tgagcacgtg agtatacgtg
     2161 attaagcaca caaaggcagc ttggagtatg tctgttatta atttcacagg tagttctggt
     2221 ccattggtga aagtttgcgg cttgcagagc acagaggccg cagaatgtgc tctagtcgag
     2281 gagaacttct agtatatcta catacctaat attattgcct tattaaaaat ggaatcccaa
     2341 caattacatc aaaatccaca ttctcttcaa aatcaattgt cctgtacttc cttgttcatg
     2401 tgtgttcaaa aacgttatat ttataggata attatactct atttctcaac aagtaattgg
     2461 ttgtttggcc gagcggtcta aggcgcctga ttcaagaaat atcttgaccg cagttaactg
     2521 tgggaatact caggtatcgt aagatgcaag agttcgaatc tcttagcaac cattattttt
     2581 ttcctcaaca taacgagaac acacaggggc gctatcgcac agaatcaaat tcgatgactg
     2641 gaaatttttt gttaatttca gaggtcgcct gacgcatata cctttttcaa ctgaaaaatt
     2701 gggagaaaaa ggaaaggtga gagcgccgga accggctttt catatagaat agagaagcgt
     2761 tcatgactaa atgcttgcat cacaatactt gaagttgaca atattattta aggacctatt
     2821 gttttttcca ataggtggtt agcaatcgtc ttactttcta acttttctta ccttttacat
     2881 ttcagcaata tatatatata tttcaaggat ataccattct aatgtctgcc cctaagaaga
     2941 tcgtcgtttt gccaggtgac cacgttggtc aagaaatcac agccgaagcc attaaggttc
     3001 ttaaagctat ttctgatgtt cgttccaatg tcaagttcga tttcgaaaat catttaattg
     3061 gtggtgctgc tatcgatgct acaggtgttc cacttccaga tgaggcgctg gaagcctcca
     3121 agaaggctga tgccgttttg ttaggtgctg tgggtggtcc taaatggggt accggtagtg
     3181 ttagacctga acaaggttta ctaaaaatcc gtaaagaact tcaattgtac gccaacttaa
     3241 gaccatgtaa ctttgcatcc gactctcttt tagacttatc tccaatcaag ccacaatttg
     3301 ctaaaggtac tgacttcgtt gttgtcagag aattagtggg aggtatttac tttggtaaga
     3361 gaaaggaaga cgatggtgat ggtgtcgctt gggatagtga acaatacacc gttccagaag
     3421 tgcaaagaat cacaagaatg gccgctttca tggccctaca acatgagcca ccattgccta
     3481 tttggtcctt ggataaagct aatgttttgg cctcttcaag attatggaga aaaactgtgg
     3541 aggaaaccat caagaacgaa ttccctacat tgaaggttca acatcaattg attgattctg
     3601 ccgccatgat cctagttaag aacccaaccc acctaaatgg tattataatc accagcaaca
     3661 tgtttggtga tatcatctcc gatgaagcct ccgttatccc aggttccttg ggtttgttgc
     3721 catctgcgtc cttggcctct ttgccagaca agaacaccgc atttggtttg tacgaaccat
     3781 gccacggttc tgctccagat ttgccaaaga ataaggtcaa ccctatcgcc actatcttgt
     3841 ctgctgcaat gatgttgaaa ttgtcattga acttgcctga agaaggtaag gccattgaag
     3901 atgcagttaa aaaggttttg gatgcaggta tcagaactgg tgatttaggt ggttccaaca
     3961 gtaccaccga agtcggtgat gctgtcgccg aagaagttaa gaaaatcctt gcttaaaaag
     4021 attctctttt tttatgatat ttgtacataa actttataaa tgaaattcat aatagaaacg
     4081 acacgaaatt acaaaatgga atatgttcat agggtagacg aaactatata cgcaatctac
     4141 atacatttat caagaaggag aaaaaggagg atgtaaagga atacaggtaa gcaaattgat
     4201 actaatggct caacgtgata aggaaaaaga attgcacttt aacattaata ttgacaagga
     4261 ggagggcacc acacaaaaag ttaggtgtaa cagaaaatca tgaaactatg attcctaatt
     4321 tatatattgg aggattttct ctaaaaaaaa aaaaatacaa caaataaaaa acactcaatg
     4381 acctgaccat ttgatggagt ttaagtcaat accttcttga accatttccc ataatggtga
     4441 aagttccctc aagaatttta ctctgtcaga aacggcctta acgacgtagt cgagatctgc
     4501 atgcaagctt ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     4561 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     4621 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc
     4681 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta
     4741 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     4801 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4861 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     4921 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     4981 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5041 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     5101 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     5161 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     5221 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     5281 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     5341 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     5401 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5461 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5521 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5581 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     5641 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     5701 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     5761 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     5821 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     5881 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     5941 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     6001 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6061 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6121 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6181 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6241 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6301 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6361 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6421 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6481 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6541 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6601 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6661 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6721 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6781 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     6841 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     6901 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     6961 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     7021 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     7081 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac atgattacga
     7141 attctgaacc agtcctaaaa cgagtaaata ggaccggcaa ttcttcaagc aataaacagg
     7201 aataccaatt attaaaagat aacttagtca gatcgtacaa taaagctttg aagaaaaatg
     7261 cgccttattc aatctttgct ataaaaaatg gcccaaaatc tcacattgga agacatttga
     7321 tgacctcatt tctttcaatg aagggcctaa cggagttgac taatgttgtg ggaaattgga
     7381 gcgataagcg tgcttctgcc gtggccagga caacgtatac tcatcagata acagcaatac
     7441 ctgatcacta cttcgcacta gtttctcggt actatgcata tgatccaata tcaaaggaaa
     7501 tgatagcatt gaaggatgag actaatccaa ttgaggagtg gcagcatata gaacagctaa
     7561 agggtagtgc tgaaggaagc atacgatacc ccgcatggaa tgggataata tcacaggagg
     7621 tactagacta cctttcatcc tacataaata gacgcatata agtacgcatt taagcataaa
     7681 cacgcactat gccgttcttc tcatgtatat atatatacag gcaacacgca gatataggtg
     7741 cgacgtgaac agtgagctgt atgtgcgcag ctcgcgttgc attttcggaa gcgctcgttt
     7801 tcggaaacgc tttgaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt
     7861 cagagcgctt ttgaaaacca aaagcgctct gaagacgcac tttcaaaaaa ccaaaaacgc
     7921 accggactgt aacgagctac taaaatattg cgaataccgc ttccacaaac attgctcaaa
     7981 agtatctctt tgctatatat ctctgtgcta tatccctata taacctaccc atccaccttt
     8041 cgctccttga acttgcatct aaactcgacc tctacatttt ttatgtttat ctctagtatt
     8101 actctttaga caaaaaaatt gtagtaagaa ctattcatag agtgaatcga aaacaatacg
     8161 aaaatgtaaa catttcctat acgtagtata tagagacaaa atagaagaaa ccgttcataa
     8221 ttttctgacc aatgaagaat catcaacgct atcactttct gttcacaaag tatgcgcaat
     8281 ccacatcggt atagaatata atcggggatg cctttatctt gaaaaaatgc acccgcagct
     8341 tcgctagtaa tcagtaaacg cgggaagtgg agtcaggctt tttttatgga agagaaaata
     8401 gacaccaaag tagccttctt ctaaccttaa cggacctaca gtgcaaaaag ttatcaagag
     8461 actgcattat agagcgcaca aaggagaaaa aaagtaatct aagatgcttt gttagaaaaa
     8521 tagcgctctc gggatgcatt tttgtagaac aaaaaagaag tatagattct ttgttggtaa
     8581 aatagcgctc tcgcgttgca tttctgttct gtaaaaatgc agctcagatt ctttgtttga
     8641 aaaattagcg ctctcgcgtt gcatttttgt tttacaaaaa tgaagcacag attcttcgtt
     8701 ggtaaaatag cgctttcgcg ttgcatttct gttctgtaaa aatgcagctc agattctttg
     8761 tttgaaaaat tagcgctctc gcgttgcatt tttgttctac aaaatgaagc acagatgctt
     8821 cgttaacaaa gatatgctat tgaagtgcaa gatggaaacg cagaaaatga accggggatg
     8881 cgacgtgcaa gattacctat gcaatagatg caatagtttc tccaggaacc gaaatacata
     8941 cattgtcttc cgtaaagcgc tagactatat attattatac aggttcaaat atactatctg
     9001 tttcagggaa aactcccagg ttcggatgtt caaaattcaa tgatgggtaa caagtacgat
     9061 cgtaaatctg taaaacagtt tgtcggatat taggctgtat ctcctcaaag cgtattcgaa
     9121 tatcattgag aagctgcagc tattcatgca actgctaaat ggtctactgc tcaattaaag
     9181 aaaacacagg cagagcaggc tgcccgggca aaagctgcag cgtcacatcg gataataatg
     9241 atggcagcca ttgtagaagt gccttttgca tttctagtct ctttctcggt ctagctagtt
     9301 ttactacatc gcgaagatag aatcttagat cacactgcct ttgctgagct ggatcaatag
     9361 agtaacaaaa gagtggtaag gcctcgttaa aggacaagga cctgagcgga agtgtatcgt
     9421 acagtagacg gagtatacta gtatagtcta tagtccgtgg aattctcatg tttgacagct
     9481 tatcatcga
//
