LOCUS       sequence-466519-        9486 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 9486)
  TITLE     Hoskins lab plasmids 2025
REFERENCE   2  (bases 1 to 9486)
  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 12bp dist
FEATURES             Location/Qualifiers
     source          1..9486
                     /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      1515..1785
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     primer_bind     complement(1865..1884)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2083..2184
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     promoter        2513..2918
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2919..4013
                     /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(4511..4528)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4511..4527)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4520..4542)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4736..4755)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4855..4877
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4915..4933)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5001..5105
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5106..5174,5175..5966)
                     /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(5324..5343)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6137..6725
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6626..6645
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6879..6896
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7013..7034
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7049..7054,7055..7072,7073..7079)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7087..7103
                     /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     7092..7114
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7111..7127
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7111..7127
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     rep_origin      7479..8821
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    7811..7858
                     /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 ccttttagac 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 ttgtttatag aggttggtac cgggttcagc gaattaatta acttccaaaa
     1081 tgaaggtcat gagtgccaat gccaatgtgg tagctgcaaa aataatgaac aatgccaaaa
     1141 atcatgtagc tgcccaacgg ggtgtaacag cgacgacaaa tgcccctgcg gtaacaagtc
     1201 tgaagaaacc aagaaatcat gctgctctgg gaaataaaac gaatagtctt taatatattc
     1261 atttaactat ttgctgtttt taatttttaa aaggagaagg aagtttaatc gacgattcta
     1321 ctcagtttga gtacacttat gtattttgtt tagatacttt gttaatttat aggtatgacc
     1381 tgcagcccaa gctggtcgac ctagataagt aatgatccgg gatctcccat gtctctactg
     1441 gtggtggtgc ttctttggaa ttattggaag gtaaggaatt gccaggtgtt gctttcttat
     1501 ccgaaaagaa ataaattgaa ttgaattgaa atcgatagat caattttttt cttttctctt
     1561 tccccatcct ttacgctaaa ataatagttt attttatttt ttgaatattt tttatttata
     1621 tacgtatata tagactatta tttatctttt aatgattatt aagattttta ttaaaaaaaa
     1681 attcgctcct cttttaatgc ctttatgcag tttttttttc ccattcgata tttctatgtt
     1741 cgggttcagc gtattttaag tttaataact cgaaaattct gcgttcgtta aagctccgga
     1801 tcgatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct
     1861 ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg
     1921 agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc
     1981 atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg
     2041 ggctgcttcc taatgcagga gtcgcataag ggagagcgtc gaaattcggt cgaaaaaaga
     2101 aaaggagagg gccaagaggg agggcattgg tgactattga gcacgtgagt atacgtgatt
     2161 aagcacacaa aggcagcttg gagtatgtct gttattaatt tcacaggtag ttctggtcca
     2221 ttggtgaaag tttgcggctt gcagagcaca gaggccgcag aatgtgctct agtcgaggag
     2281 aacttctagt atatctacat acctaatatt attgccttat taaaaatgga atcccaacaa
     2341 ttacatcaaa atccacattc tcttcaaaat caattgtcct gtacttcctt gttcatgtgt
     2401 gttcaaaaac gttatattta taggataatt atactctatt tctcaacaag taattggttg
     2461 tttggccgag cggtctaagg cgcctgattc aagaaatatc ttgaccgcag ttaactgtgg
     2521 gaatactcag gtatcgtaag atgcaagagt tcgaatctct tagcaaccat tatttttttc
     2581 ctcaacataa cgagaacaca caggggcgct atcgcacaga atcaaattcg atgactggaa
     2641 attttttgtt aatttcagag gtcgcctgac gcatatacct ttttcaactg aaaaattggg
     2701 agaaaaagga aaggtgagag cgccggaacc ggcttttcat atagaataga gaagcgttca
     2761 tgactaaatg cttgcatcac aatacttgaa gttgacaata ttatttaagg acctattgtt
     2821 ttttccaata ggtggttagc aatcgtctta ctttctaact tttcttacct tttacatttc
     2881 agcaatatat atatatattt caaggatata ccattctaat gtctgcccct aagaagatcg
     2941 tcgttttgcc aggtgaccac gttggtcaag aaatcacagc cgaagccatt aaggttctta
     3001 aagctatttc tgatgttcgt tccaatgtca agttcgattt cgaaaatcat ttaattggtg
     3061 gtgctgctat cgatgctaca ggtgttccac ttccagatga ggcgctggaa gcctccaaga
     3121 aggctgatgc cgttttgtta ggtgctgtgg gtggtcctaa atggggtacc ggtagtgtta
     3181 gacctgaaca aggtttacta aaaatccgta aagaacttca attgtacgcc aacttaagac
     3241 catgtaactt tgcatccgac tctcttttag acttatctcc aatcaagcca caatttgcta
     3301 aaggtactga cttcgttgtt gtcagagaat tagtgggagg tatttacttt ggtaagagaa
     3361 aggaagacga tggtgatggt gtcgcttggg atagtgaaca atacaccgtt ccagaagtgc
     3421 aaagaatcac aagaatggcc gctttcatgg ccctacaaca tgagccacca ttgcctattt
     3481 ggtccttgga taaagctaat gttttggcct cttcaagatt atggagaaaa actgtggagg
     3541 aaaccatcaa gaacgaattc cctacattga aggttcaaca tcaattgatt gattctgccg
     3601 ccatgatcct agttaagaac ccaacccacc taaatggtat tataatcacc agcaacatgt
     3661 ttggtgatat catctccgat gaagcctccg ttatcccagg ttccttgggt ttgttgccat
     3721 ctgcgtcctt ggcctctttg ccagacaaga acaccgcatt tggtttgtac gaaccatgcc
     3781 acggttctgc tccagatttg ccaaagaata aggtcaaccc tatcgccact atcttgtctg
     3841 ctgcaatgat gttgaaattg tcattgaact tgcctgaaga aggtaaggcc attgaagatg
     3901 cagttaaaaa ggttttggat gcaggtatca gaactggtga tttaggtggt tccaacagta
     3961 ccaccgaagt cggtgatgct gtcgccgaag aagttaagaa aatccttgct taaaaagatt
     4021 ctcttttttt atgatatttg tacataaact ttataaatga aattcataat agaaacgaca
     4081 cgaaattaca aaatggaata tgttcatagg gtagacgaaa ctatatacgc aatctacata
     4141 catttatcaa gaaggagaaa aaggaggatg taaaggaata caggtaagca aattgatact
     4201 aatggctcaa cgtgataagg aaaaagaatt gcactttaac attaatattg acaaggagga
     4261 gggcaccaca caaaaagtta ggtgtaacag aaaatcatga aactatgatt cctaatttat
     4321 atattggagg attttctcta aaaaaaaaaa aatacaacaa ataaaaaaca ctcaatgacc
     4381 tgaccatttg atggagttta agtcaatacc ttcttgaacc atttcccata atggtgaaag
     4441 ttccctcaag aattttactc tgtcagaaac ggccttaacg acgtagtcga gatctgcatg
     4501 caagcttggc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     4561 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     4621 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt
     4681 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
     4741 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
     4801 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga
     4861 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
     4921 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     4981 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     5041 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     5101 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     5161 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     5221 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     5281 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     5341 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     5401 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     5461 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     5521 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     5581 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     5641 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     5701 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     5761 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     5821 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     5881 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     5941 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     6001 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     6061 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     6121 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     6181 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     6241 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt
     6301 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     6361 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     6421 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     6481 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     6541 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     6601 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     6661 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     6721 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     6781 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     6841 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     6901 cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     6961 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga
     7021 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt
     7081 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgacatg attacgaatt
     7141 ctgaaccagt cctaaaacga gtaaatagga ccggcaattc ttcaagcaat aaacaggaat
     7201 accaattatt aaaagataac ttagtcagat cgtacaataa agctttgaag aaaaatgcgc
     7261 cttattcaat ctttgctata aaaaatggcc caaaatctca cattggaaga catttgatga
     7321 cctcatttct ttcaatgaag ggcctaacgg agttgactaa tgttgtggga aattggagcg
     7381 ataagcgtgc ttctgccgtg gccaggacaa cgtatactca tcagataaca gcaatacctg
     7441 atcactactt cgcactagtt tctcggtact atgcatatga tccaatatca aaggaaatga
     7501 tagcattgaa ggatgagact aatccaattg aggagtggca gcatatagaa cagctaaagg
     7561 gtagtgctga aggaagcata cgataccccg catggaatgg gataatatca caggaggtac
     7621 tagactacct ttcatcctac ataaatagac gcatataagt acgcatttaa gcataaacac
     7681 gcactatgcc gttcttctca tgtatatata tatacaggca acacgcagat ataggtgcga
     7741 cgtgaacagt gagctgtatg tgcgcagctc gcgttgcatt ttcggaagcg ctcgttttcg
     7801 gaaacgcttt gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcag
     7861 agcgcttttg aaaaccaaaa gcgctctgaa gacgcacttt caaaaaacca aaaacgcacc
     7921 ggactgtaac gagctactaa aatattgcga ataccgcttc cacaaacatt gctcaaaagt
     7981 atctctttgc tatatatctc tgtgctatat ccctatataa cctacccatc cacctttcgc
     8041 tccttgaact tgcatctaaa ctcgacctct acatttttta tgtttatctc tagtattact
     8101 ctttagacaa aaaaattgta gtaagaacta ttcatagagt gaatcgaaaa caatacgaaa
     8161 atgtaaacat ttcctatacg tagtatatag agacaaaata gaagaaaccg ttcataattt
     8221 tctgaccaat gaagaatcat caacgctatc actttctgtt cacaaagtat gcgcaatcca
     8281 catcggtata gaatataatc ggggatgcct ttatcttgaa aaaatgcacc cgcagcttcg
     8341 ctagtaatca gtaaacgcgg gaagtggagt caggcttttt ttatggaaga gaaaatagac
     8401 accaaagtag ccttcttcta accttaacgg acctacagtg caaaaagtta tcaagagact
     8461 gcattataga gcgcacaaag gagaaaaaaa gtaatctaag atgctttgtt agaaaaatag
     8521 cgctctcggg atgcattttt gtagaacaaa aaagaagtat agattctttg ttggtaaaat
     8581 agcgctctcg cgttgcattt ctgttctgta aaaatgcagc tcagattctt tgtttgaaaa
     8641 attagcgctc tcgcgttgca tttttgtttt acaaaaatga agcacagatt cttcgttggt
     8701 aaaatagcgc tttcgcgttg catttctgtt ctgtaaaaat gcagctcaga ttctttgttt
     8761 gaaaaattag cgctctcgcg ttgcattttt gttctacaaa atgaagcaca gatgcttcgt
     8821 taacaaagat atgctattga agtgcaagat ggaaacgcag aaaatgaacc ggggatgcga
     8881 cgtgcaagat tacctatgca atagatgcaa tagtttctcc aggaaccgaa atacatacat
     8941 tgtcttccgt aaagcgctag actatatatt attatacagg ttcaaatata ctatctgttt
     9001 cagggaaaac tcccaggttc ggatgttcaa aattcaatga tgggtaacaa gtacgatcgt
     9061 aaatctgtaa aacagtttgt cggatattag gctgtatctc ctcaaagcgt attcgaatat
     9121 cattgagaag ctgcagctat tcatgcaact gctaaatggt ctactgctca attaaagaaa
     9181 acacaggcag agcaggctgc ccgggcaaaa gctgcagcgt cacatcggat aataatgatg
     9241 gcagccattg tagaagtgcc ttttgcattt ctagtctctt tctcggtcta gctagtttta
     9301 ctacatcgcg aagatagaat cttagatcac actgcctttg ctgagctgga tcaatagagt
     9361 aacaaaagag tggtaaggcc tcgttaaagg acaaggacct gagcggaagt gtatcgtaca
     9421 gtagacggag tatactagta tagtctatag tccgtggaat tctcatgttt gacagcttat
     9481 catcga
//
