LOCUS       sequence-466531-        9516 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 9516)
  TITLE     Hoskins lab plasmids 2025
REFERENCE   2  (bases 1 to 9516)
  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 42bp dist
FEATURES             Location/Qualifiers
     source          1..9516
                     /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      1545..1815
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     primer_bind     complement(1895..1914)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2113..2214
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     promoter        2543..2948
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2949..4043
                     /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(4541..4558)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4541..4557)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4550..4572)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4766..4785)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4885..4907
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4945..4963)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5031..5135
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5136..5204,5205..5996)
                     /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(5354..5373)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6167..6755
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6656..6675
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6909..6926
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7043..7064
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7079..7084,7085..7102,7103..7109)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7117..7133
                     /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     7122..7144
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7141..7157
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7141..7157
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      7509..8851
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    7841..7888
                     /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 taacatcgaa acattgcttc attctttttg ttgctatatt atatgtttag aggttggtac
     1081 cgggttcagc gaattaatta acttccaaaa tgaaggtcat gagtgccaat gccaatgtgg
     1141 tagctgcaaa aataatgaac aatgccaaaa atcatgtagc tgcccaacgg ggtgtaacag
     1201 cgacgacaaa tgcccctgcg gtaacaagtc tgaagaaacc aagaaatcat gctgctctgg
     1261 gaaataaaac gaatagtctt taatatattc atttaactat ttgctgtttt taatttttaa
     1321 aaggagaagg aagtttaatc gacgattcta ctcagtttga gtacacttat gtattttgtt
     1381 tagatacttt gttaatttat aggtatgacc tgcagcccaa gctggtcgac ctagataagt
     1441 aatgatccgg gatctcccat gtctctactg gtggtggtgc ttctttggaa ttattggaag
     1501 gtaaggaatt gccaggtgtt gctttcttat ccgaaaagaa ataaattgaa ttgaattgaa
     1561 atcgatagat caattttttt cttttctctt tccccatcct ttacgctaaa ataatagttt
     1621 attttatttt ttgaatattt tttatttata tacgtatata tagactatta tttatctttt
     1681 aatgattatt aagattttta ttaaaaaaaa attcgctcct cttttaatgc ctttatgcag
     1741 tttttttttc ccattcgata tttctatgtt cgggttcagc gtattttaag tttaataact
     1801 cgaaaattct gcgttcgtta aagctccgga tcgatcctct acgccggacg catcgtggcc
     1861 ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg
     1921 gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca
     1981 ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg
     2041 gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag
     2101 ggagagcgtc gaaattcggt cgaaaaaaga aaaggagagg gccaagaggg agggcattgg
     2161 tgactattga gcacgtgagt atacgtgatt aagcacacaa aggcagcttg gagtatgtct
     2221 gttattaatt tcacaggtag ttctggtcca ttggtgaaag tttgcggctt gcagagcaca
     2281 gaggccgcag aatgtgctct agtcgaggag aacttctagt atatctacat acctaatatt
     2341 attgccttat taaaaatgga atcccaacaa ttacatcaaa atccacattc tcttcaaaat
     2401 caattgtcct gtacttcctt gttcatgtgt gttcaaaaac gttatattta taggataatt
     2461 atactctatt tctcaacaag taattggttg tttggccgag cggtctaagg cgcctgattc
     2521 aagaaatatc ttgaccgcag ttaactgtgg gaatactcag gtatcgtaag atgcaagagt
     2581 tcgaatctct tagcaaccat tatttttttc ctcaacataa cgagaacaca caggggcgct
     2641 atcgcacaga atcaaattcg atgactggaa attttttgtt aatttcagag gtcgcctgac
     2701 gcatatacct ttttcaactg aaaaattggg agaaaaagga aaggtgagag cgccggaacc
     2761 ggcttttcat atagaataga gaagcgttca tgactaaatg cttgcatcac aatacttgaa
     2821 gttgacaata ttatttaagg acctattgtt ttttccaata ggtggttagc aatcgtctta
     2881 ctttctaact tttcttacct tttacatttc agcaatatat atatatattt caaggatata
     2941 ccattctaat gtctgcccct aagaagatcg tcgttttgcc aggtgaccac gttggtcaag
     3001 aaatcacagc cgaagccatt aaggttctta aagctatttc tgatgttcgt tccaatgtca
     3061 agttcgattt cgaaaatcat ttaattggtg gtgctgctat cgatgctaca ggtgttccac
     3121 ttccagatga ggcgctggaa gcctccaaga aggctgatgc cgttttgtta ggtgctgtgg
     3181 gtggtcctaa atggggtacc ggtagtgtta gacctgaaca aggtttacta aaaatccgta
     3241 aagaacttca attgtacgcc aacttaagac catgtaactt tgcatccgac tctcttttag
     3301 acttatctcc aatcaagcca caatttgcta aaggtactga cttcgttgtt gtcagagaat
     3361 tagtgggagg tatttacttt ggtaagagaa aggaagacga tggtgatggt gtcgcttggg
     3421 atagtgaaca atacaccgtt ccagaagtgc aaagaatcac aagaatggcc gctttcatgg
     3481 ccctacaaca tgagccacca ttgcctattt ggtccttgga taaagctaat gttttggcct
     3541 cttcaagatt atggagaaaa actgtggagg aaaccatcaa gaacgaattc cctacattga
     3601 aggttcaaca tcaattgatt gattctgccg ccatgatcct agttaagaac ccaacccacc
     3661 taaatggtat tataatcacc agcaacatgt ttggtgatat catctccgat gaagcctccg
     3721 ttatcccagg ttccttgggt ttgttgccat ctgcgtcctt ggcctctttg ccagacaaga
     3781 acaccgcatt tggtttgtac gaaccatgcc acggttctgc tccagatttg ccaaagaata
     3841 aggtcaaccc tatcgccact atcttgtctg ctgcaatgat gttgaaattg tcattgaact
     3901 tgcctgaaga aggtaaggcc attgaagatg cagttaaaaa ggttttggat gcaggtatca
     3961 gaactggtga tttaggtggt tccaacagta ccaccgaagt cggtgatgct gtcgccgaag
     4021 aagttaagaa aatccttgct taaaaagatt ctcttttttt atgatatttg tacataaact
     4081 ttataaatga aattcataat agaaacgaca cgaaattaca aaatggaata tgttcatagg
     4141 gtagacgaaa ctatatacgc aatctacata catttatcaa gaaggagaaa aaggaggatg
     4201 taaaggaata caggtaagca aattgatact aatggctcaa cgtgataagg aaaaagaatt
     4261 gcactttaac attaatattg acaaggagga gggcaccaca caaaaagtta ggtgtaacag
     4321 aaaatcatga aactatgatt cctaatttat atattggagg attttctcta aaaaaaaaaa
     4381 aatacaacaa ataaaaaaca ctcaatgacc tgaccatttg atggagttta agtcaatacc
     4441 ttcttgaacc atttcccata atggtgaaag ttccctcaag aattttactc tgtcagaaac
     4501 ggccttaacg acgtagtcga gatctgcatg caagcttggc actggccgtc gttttacaac
     4561 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     4621 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     4681 gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4741 cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     4801 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     4861 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     4921 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     4981 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5041 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5101 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5161 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5221 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5281 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5341 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     5401 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5461 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     5521 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     5581 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     5641 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     5701 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     5761 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     5821 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     5881 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     5941 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6001 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6061 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6121 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6181 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6241 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6301 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6361 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6421 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6481 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6541 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6601 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6661 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     6721 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     6781 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     6841 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     6901 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc
     6961 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     7021 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
     7081 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
     7141 caggaaacag ctatgacatg attacgaatt ctgaaccagt cctaaaacga gtaaatagga
     7201 ccggcaattc ttcaagcaat aaacaggaat accaattatt aaaagataac ttagtcagat
     7261 cgtacaataa agctttgaag aaaaatgcgc cttattcaat ctttgctata aaaaatggcc
     7321 caaaatctca cattggaaga catttgatga cctcatttct ttcaatgaag ggcctaacgg
     7381 agttgactaa tgttgtggga aattggagcg ataagcgtgc ttctgccgtg gccaggacaa
     7441 cgtatactca tcagataaca gcaatacctg atcactactt cgcactagtt tctcggtact
     7501 atgcatatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg
     7561 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg
     7621 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac
     7681 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata
     7741 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc
     7801 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt
     7861 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa
     7921 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga
     7981 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat
     8041 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct
     8101 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta
     8161 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag
     8221 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc
     8281 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct
     8341 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt
     8401 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg
     8461 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa
     8521 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa
     8581 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta
     8641 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt
     8701 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt
     8761 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt
     8821 gttctacaaa atgaagcaca gatgcttcgt taacaaagat atgctattga agtgcaagat
     8881 ggaaacgcag aaaatgaacc ggggatgcga cgtgcaagat tacctatgca atagatgcaa
     8941 tagtttctcc aggaaccgaa atacatacat tgtcttccgt aaagcgctag actatatatt
     9001 attatacagg ttcaaatata ctatctgttt cagggaaaac tcccaggttc ggatgttcaa
     9061 aattcaatga tgggtaacaa gtacgatcgt aaatctgtaa aacagtttgt cggatattag
     9121 gctgtatctc ctcaaagcgt attcgaatat cattgagaag ctgcagctat tcatgcaact
     9181 gctaaatggt ctactgctca attaaagaaa acacaggcag agcaggctgc ccgggcaaaa
     9241 gctgcagcgt cacatcggat aataatgatg gcagccattg tagaagtgcc ttttgcattt
     9301 ctagtctctt tctcggtcta gctagtttta ctacatcgcg aagatagaat cttagatcac
     9361 actgcctttg ctgagctgga tcaatagagt aacaaaagag tggtaaggcc tcgttaaagg
     9421 acaaggacct gagcggaagt gtatcgtaca gtagacggag tatactagta tagtctatag
     9481 tccgtggaat tctcatgttt gacagcttat catcga
//
