LOCUS       sequence-466539-        9501 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 9501)
  TITLE     Hoskins lab plasmids 2025
REFERENCE   2  (bases 1 to 9501)
  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 27bp dist
FEATURES             Location/Qualifiers
     source          1..9501
                     /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      1530..1800
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     primer_bind     complement(1880..1899)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2098..2199
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     promoter        2528..2933
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2934..4028
                     /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(4526..4543)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4526..4542)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4535..4557)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4751..4770)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4870..4892
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4930..4948)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5016..5120
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5121..5189,5190..5981)
                     /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(5339..5358)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6152..6740
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6641..6660
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6894..6911
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7028..7049
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7064..7069,7070..7087,7088..7094)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7102..7118
                     /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     7107..7129
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7126..7142
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7126..7142
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      7494..8836
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    7826..7873
                     /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 taacatcgtt cttctttccg attatatgtt tatagaggtt ggtaccgggt tcagcgaatt
     1081 aattaacttc caaaatgaag gtcatgagtg ccaatgccaa tgtggtagct gcaaaaataa
     1141 tgaacaatgc caaaaatcat gtagctgccc aacggggtgt aacagcgacg acaaatgccc
     1201 ctgcggtaac aagtctgaag aaaccaagaa atcatgctgc tctgggaaat aaaacgaata
     1261 gtctttaata tattcattta actatttgct gtttttaatt tttaaaagga gaaggaagtt
     1321 taatcgacga ttctactcag tttgagtaca cttatgtatt ttgtttagat actttgttaa
     1381 tttataggta tgacctgcag cccaagctgg tcgacctaga taagtaatga tccgggatct
     1441 cccatgtctc tactggtggt ggtgcttctt tggaattatt ggaaggtaag gaattgccag
     1501 gtgttgcttt cttatccgaa aagaaataaa ttgaattgaa ttgaaatcga tagatcaatt
     1561 tttttctttt ctctttcccc atcctttacg ctaaaataat agtttatttt attttttgaa
     1621 tattttttat ttatatacgt atatatagac tattatttat cttttaatga ttattaagat
     1681 ttttattaaa aaaaaattcg ctcctctttt aatgccttta tgcagttttt ttttcccatt
     1741 cgatatttct atgttcgggt tcagcgtatt ttaagtttaa taactcgaaa attctgcgtt
     1801 cgttaaagct ccggatcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc
     1861 acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc
     1921 cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg
     1981 ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc
     2041 ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgaaat
     2101 tcggtcgaaa aaagaaaagg agagggccaa gagggagggc attggtgact attgagcacg
     2161 tgagtatacg tgattaagca cacaaaggca gcttggagta tgtctgttat taatttcaca
     2221 ggtagttctg gtccattggt gaaagtttgc ggcttgcaga gcacagaggc cgcagaatgt
     2281 gctctagtcg aggagaactt ctagtatatc tacataccta atattattgc cttattaaaa
     2341 atggaatccc aacaattaca tcaaaatcca cattctcttc aaaatcaatt gtcctgtact
     2401 tccttgttca tgtgtgttca aaaacgttat atttatagga taattatact ctatttctca
     2461 acaagtaatt ggttgtttgg ccgagcggtc taaggcgcct gattcaagaa atatcttgac
     2521 cgcagttaac tgtgggaata ctcaggtatc gtaagatgca agagttcgaa tctcttagca
     2581 accattattt ttttcctcaa cataacgaga acacacaggg gcgctatcgc acagaatcaa
     2641 attcgatgac tggaaatttt ttgttaattt cagaggtcgc ctgacgcata tacctttttc
     2701 aactgaaaaa ttgggagaaa aaggaaaggt gagagcgccg gaaccggctt ttcatataga
     2761 atagagaagc gttcatgact aaatgcttgc atcacaatac ttgaagttga caatattatt
     2821 taaggaccta ttgttttttc caataggtgg ttagcaatcg tcttactttc taacttttct
     2881 taccttttac atttcagcaa tatatatata tatttcaagg atataccatt ctaatgtctg
     2941 cccctaagaa gatcgtcgtt ttgccaggtg accacgttgg tcaagaaatc acagccgaag
     3001 ccattaaggt tcttaaagct atttctgatg ttcgttccaa tgtcaagttc gatttcgaaa
     3061 atcatttaat tggtggtgct gctatcgatg ctacaggtgt tccacttcca gatgaggcgc
     3121 tggaagcctc caagaaggct gatgccgttt tgttaggtgc tgtgggtggt cctaaatggg
     3181 gtaccggtag tgttagacct gaacaaggtt tactaaaaat ccgtaaagaa cttcaattgt
     3241 acgccaactt aagaccatgt aactttgcat ccgactctct tttagactta tctccaatca
     3301 agccacaatt tgctaaaggt actgacttcg ttgttgtcag agaattagtg ggaggtattt
     3361 actttggtaa gagaaaggaa gacgatggtg atggtgtcgc ttgggatagt gaacaataca
     3421 ccgttccaga agtgcaaaga atcacaagaa tggccgcttt catggcccta caacatgagc
     3481 caccattgcc tatttggtcc ttggataaag ctaatgtttt ggcctcttca agattatgga
     3541 gaaaaactgt ggaggaaacc atcaagaacg aattccctac attgaaggtt caacatcaat
     3601 tgattgattc tgccgccatg atcctagtta agaacccaac ccacctaaat ggtattataa
     3661 tcaccagcaa catgtttggt gatatcatct ccgatgaagc ctccgttatc ccaggttcct
     3721 tgggtttgtt gccatctgcg tccttggcct ctttgccaga caagaacacc gcatttggtt
     3781 tgtacgaacc atgccacggt tctgctccag atttgccaaa gaataaggtc aaccctatcg
     3841 ccactatctt gtctgctgca atgatgttga aattgtcatt gaacttgcct gaagaaggta
     3901 aggccattga agatgcagtt aaaaaggttt tggatgcagg tatcagaact ggtgatttag
     3961 gtggttccaa cagtaccacc gaagtcggtg atgctgtcgc cgaagaagtt aagaaaatcc
     4021 ttgcttaaaa agattctctt tttttatgat atttgtacat aaactttata aatgaaattc
     4081 ataatagaaa cgacacgaaa ttacaaaatg gaatatgttc atagggtaga cgaaactata
     4141 tacgcaatct acatacattt atcaagaagg agaaaaagga ggatgtaaag gaatacaggt
     4201 aagcaaattg atactaatgg ctcaacgtga taaggaaaaa gaattgcact ttaacattaa
     4261 tattgacaag gaggagggca ccacacaaaa agttaggtgt aacagaaaat catgaaacta
     4321 tgattcctaa tttatatatt ggaggatttt ctctaaaaaa aaaaaaatac aacaaataaa
     4381 aaacactcaa tgacctgacc atttgatgga gtttaagtca ataccttctt gaaccatttc
     4441 ccataatggt gaaagttccc tcaagaattt tactctgtca gaaacggcct taacgacgta
     4501 gtcgagatct gcatgcaagc ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     4561 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     4621 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     4681 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     4741 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     4801 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4861 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4921 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     4981 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5041 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5101 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5161 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5221 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5281 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5341 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5401 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5461 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5521 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5581 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5641 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     5701 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5761 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     5821 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     5881 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     5941 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6001 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6061 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6121 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6181 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6241 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6301 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6361 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6421 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6481 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6541 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6601 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6661 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6721 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     6781 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     6841 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     6901 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     6961 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     7021 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     7081 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     7141 acatgattac gaattctgaa ccagtcctaa aacgagtaaa taggaccggc aattcttcaa
     7201 gcaataaaca ggaataccaa ttattaaaag ataacttagt cagatcgtac aataaagctt
     7261 tgaagaaaaa tgcgccttat tcaatctttg ctataaaaaa tggcccaaaa tctcacattg
     7321 gaagacattt gatgacctca tttctttcaa tgaagggcct aacggagttg actaatgttg
     7381 tgggaaattg gagcgataag cgtgcttctg ccgtggccag gacaacgtat actcatcaga
     7441 taacagcaat acctgatcac tacttcgcac tagtttctcg gtactatgca tatgatccaa
     7501 tatcaaagga aatgatagca ttgaaggatg agactaatcc aattgaggag tggcagcata
     7561 tagaacagct aaagggtagt gctgaaggaa gcatacgata ccccgcatgg aatgggataa
     7621 tatcacagga ggtactagac tacctttcat cctacataaa tagacgcata taagtacgca
     7681 tttaagcata aacacgcact atgccgttct tctcatgtat atatatatac aggcaacacg
     7741 cagatatagg tgcgacgtga acagtgagct gtatgtgcgc agctcgcgtt gcattttcgg
     7801 aagcgctcgt tttcggaaac gctttgaagt tcctattccg aagttcctat tctctagaaa
     7861 gtataggaac ttcagagcgc ttttgaaaac caaaagcgct ctgaagacgc actttcaaaa
     7921 aaccaaaaac gcaccggact gtaacgagct actaaaatat tgcgaatacc gcttccacaa
     7981 acattgctca aaagtatctc tttgctatat atctctgtgc tatatcccta tataacctac
     8041 ccatccacct ttcgctcctt gaacttgcat ctaaactcga cctctacatt ttttatgttt
     8101 atctctagta ttactcttta gacaaaaaaa ttgtagtaag aactattcat agagtgaatc
     8161 gaaaacaata cgaaaatgta aacatttcct atacgtagta tatagagaca aaatagaaga
     8221 aaccgttcat aattttctga ccaatgaaga atcatcaacg ctatcacttt ctgttcacaa
     8281 agtatgcgca atccacatcg gtatagaata taatcgggga tgcctttatc ttgaaaaaat
     8341 gcacccgcag cttcgctagt aatcagtaaa cgcgggaagt ggagtcaggc tttttttatg
     8401 gaagagaaaa tagacaccaa agtagccttc ttctaacctt aacggaccta cagtgcaaaa
     8461 agttatcaag agactgcatt atagagcgca caaaggagaa aaaaagtaat ctaagatgct
     8521 ttgttagaaa aatagcgctc tcgggatgca tttttgtaga acaaaaaaga agtatagatt
     8581 ctttgttggt aaaatagcgc tctcgcgttg catttctgtt ctgtaaaaat gcagctcaga
     8641 ttctttgttt gaaaaattag cgctctcgcg ttgcattttt gttttacaaa aatgaagcac
     8701 agattcttcg ttggtaaaat agcgctttcg cgttgcattt ctgttctgta aaaatgcagc
     8761 tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgttct acaaaatgaa
     8821 gcacagatgc ttcgttaaca aagatatgct attgaagtgc aagatggaaa cgcagaaaat
     8881 gaaccgggga tgcgacgtgc aagattacct atgcaataga tgcaatagtt tctccaggaa
     8941 ccgaaataca tacattgtct tccgtaaagc gctagactat atattattat acaggttcaa
     9001 atatactatc tgtttcaggg aaaactccca ggttcggatg ttcaaaattc aatgatgggt
     9061 aacaagtacg atcgtaaatc tgtaaaacag tttgtcggat attaggctgt atctcctcaa
     9121 agcgtattcg aatatcattg agaagctgca gctattcatg caactgctaa atggtctact
     9181 gctcaattaa agaaaacaca ggcagagcag gctgcccggg caaaagctgc agcgtcacat
     9241 cggataataa tgatggcagc cattgtagaa gtgccttttg catttctagt ctctttctcg
     9301 gtctagctag ttttactaca tcgcgaagat agaatcttag atcacactgc ctttgctgag
     9361 ctggatcaat agagtaacaa aagagtggta aggcctcgtt aaaggacaag gacctgagcg
     9421 gaagtgtatc gtacagtaga cggagtatac tagtatagtc tatagtccgt ggaattctca
     9481 tgtttgacag cttatcatcg a
//
