LOCUS       sequence-466054-        9523 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 9523)
  TITLE     Hoskins lab plasmids 2025
REFERENCE   2  (bases 1 to 9523)
  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 50bp dist
FEATURES             Location/Qualifiers
     source          1..9523
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      721..2063
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(1684..1731)
                     /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)."
     primer_bind     complement(2415..2431)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2415..2431)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2428..2450)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2439..2455
                     /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)."
     promoter        complement(join(2463..2469,2470..2487,2488..2493))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2508..2529
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2646..2663)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2817..3405)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2897..2916)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3576..4367,4368..4436))
                     /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     4199..4218
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4437..4541)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4609..4627
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4665..4687)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4787..4806
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5000..5022
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5014..5031
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5015..5031
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(5529..6623)
                     /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"
     promoter        complement(6624..7029)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     promoter        complement(7358..7459)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     7658..7677
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     terminator      complement(7757..8027)
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     promoter        complement(join(8910..9523,1..30))
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     complement(8994..9017)
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
ORIGIN
        1 tattctttga aatggcagta ttgataatga taaactcgac ctgcagccaa gcttatcgat
       61 gataagctgt caaacatgag aattccacgg actatagact atactagtat actccgtcta
      121 ctgtacgata cacttccgct caggtccttg tcctttaacg aggccttacc actcttttgt
      181 tactctattg atccagctca gcaaaggcag tgtgatctaa gattctatct tcgcgatgta
      241 gtaaaactag ctagaccgag aaagagacta gaaatgcaaa aggcacttct acaatggctg
      301 ccatcattat tatccgatgt gacgctgcag cttttgcccg ggcagcctgc tctgcctgtg
      361 ttttctttaa ttgagcagta gaccatttag cagttgcatg aatagctgca gcttctcaat
      421 gatattcgaa tacgctttga ggagatacag cctaatatcc gacaaactgt tttacagatt
      481 tacgatcgta cttgttaccc atcattgaat tttgaacatc cgaacctggg agttttccct
      541 gaaacagata gtatatttga acctgtataa taatatatag tctagcgctt tacggaagac
      601 aatgtatgta tttcggttcc tggagaaact attgcatcta ttgcataggt aatcttgcac
      661 gtcgcatccc cggttcattt tctgcgtttc catcttgcac ttcaatagca tatctttgtt
      721 aacgaagcat ctgtgcttca ttttgtagaa caaaaatgca acgcgagagc gctaattttt
      781 caaacaaaga atctgagctg catttttaca gaacagaaat gcaacgcgaa agcgctattt
      841 taccaacgaa gaatctgtgc ttcatttttg taaaacaaaa atgcaacgcg agagcgctaa
      901 tttttcaaac aaagaatctg agctgcattt ttacagaaca gaaatgcaac gcgagagcgc
      961 tattttacca acaaagaatc tatacttctt ttttgttcta caaaaatgca tcccgagagc
     1021 gctatttttc taacaaagca tcttagatta ctttttttct cctttgtgcg ctctataatg
     1081 cagtctcttg ataacttttt gcactgtagg tccgttaagg ttagaagaag gctactttgg
     1141 tgtctatttt ctcttccata aaaaaagcct gactccactt cccgcgttta ctgattacta
     1201 gcgaagctgc gggtgcattt tttcaagata aaggcatccc cgattatatt ctataccgat
     1261 gtggattgcg catactttgt gaacagaaag tgatagcgtt gatgattctt cattggtcag
     1321 aaaattatga acggtttctt ctattttgtc tctatatact acgtatagga aatgtttaca
     1381 ttttcgtatt gttttcgatt cactctatga atagttctta ctacaatttt tttgtctaaa
     1441 gagtaatact agagataaac ataaaaaatg tagaggtcga gtttagatgc aagttcaagg
     1501 agcgaaaggt ggatgggtag gttatatagg gatatagcac agagatatat agcaaagaga
     1561 tacttttgag caatgtttgt ggaagcggta ttcgcaatat tttagtagct cgttacagtc
     1621 cggtgcgttt ttggtttttt gaaagtgcgt cttcagagcg cttttggttt tcaaaagcgc
     1681 tctgaagttc ctatactttc tagagaatag gaacttcgga ataggaactt caaagcgttt
     1741 ccgaaaacga gcgcttccga aaatgcaacg cgagctgcgc acatacagct cactgttcac
     1801 gtcgcaccta tatctgcgtg ttgcctgtat atatatatac atgagaagaa cggcatagtg
     1861 cgtgtttatg cttaaatgcg tacttatatg cgtctattta tgtaggatga aaggtagtct
     1921 agtacctcct gtgatattat cccattccat gcggggtatc gtatgcttcc ttcagcacta
     1981 ccctttagct gttctatatg ctgccactcc tcaattggat tagtctcatc cttcaatgct
     2041 atcatttcct ttgatattgg atcatatgca tagtaccgag aaactagtgc gaagtagtga
     2101 tcaggtattg ctgttatctg atgagtatac gttgtcctgg ccacggcaga agcacgctta
     2161 tcgctccaat ttcccacaac attagtcaac tccgttaggc ccttcattga aagaaatgag
     2221 gtcatcaaat gtcttccaat gtgagatttt gggccatttt ttatagcaaa gattgaataa
     2281 ggcgcatttt tcttcaaagc tttattgtac gatctgacta agttatcttt taataattgg
     2341 tattcctgtt tattgcttga agaattgccg gtcctattta ctcgttttag gactggttca
     2401 gaattcgtaa tcatgtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     2461 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     2521 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     2581 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     2641 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2701 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2761 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2821 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2881 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2941 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3001 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3061 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3121 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3181 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3241 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3301 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3361 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3421 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     3481 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     3541 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     3601 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3661 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     3721 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3781 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3841 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3901 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3961 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4021 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4081 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4141 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     4201 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     4261 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     4321 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     4381 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     4441 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4501 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     4561 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     4621 acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     4681 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     4741 ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag
     4801 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     4861 taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg
     4921 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt
     4981 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgccaagctt
     5041 gcatgcagat ctcgactacg tcgttaaggc cgtttctgac agagtaaaat tcttgaggga
     5101 actttcacca ttatgggaaa tggttcaaga aggtattgac ttaaactcca tcaaatggtc
     5161 aggtcattga gtgtttttta tttgttgtat tttttttttt ttagagaaaa tcctccaata
     5221 tataaattag gaatcatagt ttcatgattt tctgttacac ctaacttttt gtgtggtgcc
     5281 ctcctccttg tcaatattaa tgttaaagtg caattctttt tccttatcac gttgagccat
     5341 tagtatcaat ttgcttacct gtattccttt acatcctcct ttttctcctt cttgataaat
     5401 gtatgtagat tgcgtatata gtttcgtcta ccctatgaac atattccatt ttgtaatttc
     5461 gtgtcgtttc tattatgaat ttcatttata aagtttatgt acaaatatca taaaaaaaga
     5521 gaatcttttt aagcaaggat tttcttaact tcttcggcga cagcatcacc gacttcggtg
     5581 gtactgttgg aaccacctaa atcaccagtt ctgatacctg catccaaaac ctttttaact
     5641 gcatcttcaa tggccttacc ttcttcaggc aagttcaatg acaatttcaa catcattgca
     5701 gcagacaaga tagtggcgat agggttgacc ttattctttg gcaaatctgg agcagaaccg
     5761 tggcatggtt cgtacaaacc aaatgcggtg ttcttgtctg gcaaagaggc caaggacgca
     5821 gatggcaaca aacccaagga acctgggata acggaggctt catcggagat gatatcacca
     5881 aacatgttgc tggtgattat aataccattt aggtgggttg ggttcttaac taggatcatg
     5941 gcggcagaat caatcaattg atgttgaacc ttcaatgtag ggaattcgtt cttgatggtt
     6001 tcctccacag tttttctcca taatcttgaa gaggccaaaa cattagcttt atccaaggac
     6061 caaataggca atggtggctc atgttgtagg gccatgaaag cggccattct tgtgattctt
     6121 tgcacttctg gaacggtgta ttgttcacta tcccaagcga caccatcacc atcgtcttcc
     6181 tttctcttac caaagtaaat acctcccact aattctctga caacaacgaa gtcagtacct
     6241 ttagcaaatt gtggcttgat tggagataag tctaaaagag agtcggatgc aaagttacat
     6301 ggtcttaagt tggcgtacaa ttgaagttct ttacggattt ttagtaaacc ttgttcaggt
     6361 ctaacactac cggtacccca tttaggacca cccacagcac ctaacaaaac ggcatcagcc
     6421 ttcttggagg cttccagcgc ctcatctgga agtggaacac ctgtagcatc gatagcagca
     6481 ccaccaatta aatgattttc gaaatcgaac ttgacattgg aacgaacatc agaaatagct
     6541 ttaagaacct taatggcttc ggctgtgatt tcttgaccaa cgtggtcacc tggcaaaacg
     6601 acgatcttct taggggcaga cattagaatg gtatatcctt gaaatatata tatatattgc
     6661 tgaaatgtaa aaggtaagaa aagttagaaa gtaagacgat tgctaaccac ctattggaaa
     6721 aaacaatagg tccttaaata atattgtcaa cttcaagtat tgtgatgcaa gcatttagtc
     6781 atgaacgctt ctctattcta tatgaaaagc cggttccggc gctctcacct ttcctttttc
     6841 tcccaatttt tcagttgaaa aaggtatatg cgtcaggcga cctctgaaat taacaaaaaa
     6901 tttccagtca tcgaatttga ttctgtgcga tagcgcccct gtgtgttctc gttatgttga
     6961 ggaaaaaaat aatggttgct aagagattcg aactcttgca tcttacgata cctgagtatt
     7021 cccacagtta actgcggtca agatatttct tgaatcaggc gccttagacc gctcggccaa
     7081 acaaccaatt acttgttgag aaatagagta taattatcct ataaatataa cgtttttgaa
     7141 cacacatgaa caaggaagta caggacaatt gattttgaag agaatgtgga ttttgatgta
     7201 attgttggga ttccattttt aataaggcaa taatattagg tatgtagata tactagaagt
     7261 tctcctcgac tagagcacat tctgcggcct ctgtgctctg caagccgcaa actttcacca
     7321 atggaccaga actacctgtg aaattaataa cagacatact ccaagctgcc tttgtgtgct
     7381 taatcacgta tactcacgtg ctcaatagtc accaatgccc tccctcttgg ccctctcctt
     7441 ttcttttttc gaccgaattt cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     7501 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     7561 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     7621 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     7681 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     7741 atccggagct ttaacgaacg cagaattttc gagttattaa acttaaaata cgctgaaccc
     7801 gaacatagaa atatcgaatg ggaaaaaaaa actgcataaa ggcattaaaa gaggagcgaa
     7861 ttttttttta ataaaaatct taataatcat taaaagataa ataatagtct atatatacgt
     7921 atataaataa aaaatattca aaaaataaaa taaactatta ttttagcgta aaggatgggg
     7981 aaagagaaaa gaaaaaaatt gatctatcga tttcaattca attcaattta tttcttttcg
     8041 gataagaaag caacacctgg caattcctta ccttccaata attccaaaga agcaccacca
     8101 ccagtagaga catgggagat cccggatcat tacttatcta ggtcgaccag cttgggctgc
     8161 aggtcatacc tataaattaa caaagtatct aaacaaaata cataagtgta ctcaaactga
     8221 gtagaatcgt cgattaaact tccttctcct tttaaaaatt aaaaacagca aatagttaaa
     8281 tgaatatatt aaagactatt cgttttattt cccagagcag catgatttct tggtttcttc
     8341 agacttgtta ccgcaggggc atttgtcgtc gctgttacac cccgttgggc agctacatga
     8401 tttttggcat tgttcattat ttttgcagct accacattgg cattggcact catgaccttc
     8461 attttggaag ttaattaatt cgctgaaccc ggtaccaacc tctaaacata taatatagca
     8521 acaaaaagaa tgaagcaatc gtttgttgtt tcgatgttag tacatgagac acagtagcaa
     8581 ataaaagaag aatatcacta tcacttatca cgaaaatatt tcagtttttt ttttttccaa
     8641 atttcaagcc cctatttatt ccaataatat cgtggttatt acagatcagt caatatagga
     8701 ggttatggga gagtgaaaaa tagtaaaaaa aggtaaaaga gaaatctctc gagcaattgg
     8761 gaccgtgcaa ttcttcttac agttaaatgg gatggtgcaa gcgctagaac ataccagaat
     8821 ccattgttaa ttcagtaaat tttcgatctt gggaagaaaa aagcagtaag cgtgaaaaat
     8881 ctaaaagggg cgagctcgcc ggggatccgt cgaaactaag ttcttggtgt tttaaaacta
     8941 aaaaaaagac taactataaa agtagaattt aagaagttta agaaatagat ttacagaatt
     9001 acaatcaata cctaccgtct ttatatactt attagtcaag taggggaata atttcaggga
     9061 actggtttca accttttttt tcagcttttt ccaaatcaga gagagcagaa ggtaatagaa
     9121 ggtgtaagaa aatgagatag atacatgcgt gggtcaattg ccttgtgtca tcatttactc
     9181 caggcaggtt gcatcactcc attgaggttg tgcccgtttt ttgcctgttt gtgcccctgt
     9241 tctctgtagt tgcgctaaga gaatggacct atgaactgat ggttggtgaa gaaaacaata
     9301 ttttggtgct gggattcttt ttttttctgg atgccagctt aaaaagcggg ctccattata
     9361 tttagtggat gccaggaata aactgttcac ccagacacct acgatgttat atattctgtg
     9421 taacccgccc cctattttgg gcatgtacgg gttacagcag aattaaaagg ctaatttttt
     9481 gactaaataa agttaggaaa atcactacta ttaattattt acg
//
