LOCUS       sequence-466051-        9518 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 9518)
  TITLE     Hoskins lab plasmids 2025
REFERENCE   2  (bases 1 to 9518)
  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 46bp dist
FEATURES             Location/Qualifiers
     source          1..9518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      707..2049
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    complement(1670..1717)
                     /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(2401..2417)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2401..2417)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2414..2436)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2425..2441
                     /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(2449..2455,2456..2473,2474..2479))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2494..2515
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2632..2649)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2803..3391)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2883..2902)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3562..4353,4354..4422))
                     /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     4185..4204
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4423..4527)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4595..4613
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4651..4673)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4773..4792
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4986..5008
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5000..5017
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5001..5017
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(5515..6609)
                     /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(6610..7015)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     promoter        complement(7344..7445)
                     /gene="S. cerevisiae TRP1"
                     /label=TRP1 promoter
     primer_bind     7644..7663
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     terminator      complement(7743..8013)
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     promoter        complement(join(8891..9518,1..16))
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     complement(8975..8998)
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
ORIGIN
        1 gcagtattga taatgataaa ctcgacctgc agccaagctt atcgatgata agctgtcaaa
       61 catgagaatt ccacggacta tagactatac tagtatactc cgtctactgt acgatacact
      121 tccgctcagg tccttgtcct ttaacgaggc cttaccactc ttttgttact ctattgatcc
      181 agctcagcaa aggcagtgtg atctaagatt ctatcttcgc gatgtagtaa aactagctag
      241 accgagaaag agactagaaa tgcaaaaggc acttctacaa tggctgccat cattattatc
      301 cgatgtgacg ctgcagcttt tgcccgggca gcctgctctg cctgtgtttt ctttaattga
      361 gcagtagacc atttagcagt tgcatgaata gctgcagctt ctcaatgata ttcgaatacg
      421 ctttgaggag atacagccta atatccgaca aactgtttta cagatttacg atcgtacttg
      481 ttacccatca ttgaattttg aacatccgaa cctgggagtt ttccctgaaa cagatagtat
      541 atttgaacct gtataataat atatagtcta gcgctttacg gaagacaatg tatgtatttc
      601 ggttcctgga gaaactattg catctattgc ataggtaatc ttgcacgtcg catccccggt
      661 tcattttctg cgtttccatc ttgcacttca atagcatatc tttgttaacg aagcatctgt
      721 gcttcatttt gtagaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct
      781 gagctgcatt tttacagaac agaaatgcaa cgcgaaagcg ctattttacc aacgaagaat
      841 ctgtgcttca tttttgtaaa acaaaaatgc aacgcgagag cgctaatttt tcaaacaaag
      901 aatctgagct gcatttttac agaacagaaa tgcaacgcga gagcgctatt ttaccaacaa
      961 agaatctata cttctttttt gttctacaaa aatgcatccc gagagcgcta tttttctaac
     1021 aaagcatctt agattacttt ttttctcctt tgtgcgctct ataatgcagt ctcttgataa
     1081 ctttttgcac tgtaggtccg ttaaggttag aagaaggcta ctttggtgtc tattttctct
     1141 tccataaaaa aagcctgact ccacttcccg cgtttactga ttactagcga agctgcgggt
     1201 gcattttttc aagataaagg catccccgat tatattctat accgatgtgg attgcgcata
     1261 ctttgtgaac agaaagtgat agcgttgatg attcttcatt ggtcagaaaa ttatgaacgg
     1321 tttcttctat tttgtctcta tatactacgt ataggaaatg tttacatttt cgtattgttt
     1381 tcgattcact ctatgaatag ttcttactac aatttttttg tctaaagagt aatactagag
     1441 ataaacataa aaaatgtaga ggtcgagttt agatgcaagt tcaaggagcg aaaggtggat
     1501 gggtaggtta tatagggata tagcacagag atatatagca aagagatact tttgagcaat
     1561 gtttgtggaa gcggtattcg caatatttta gtagctcgtt acagtccggt gcgtttttgg
     1621 ttttttgaaa gtgcgtcttc agagcgcttt tggttttcaa aagcgctctg aagttcctat
     1681 actttctaga gaataggaac ttcggaatag gaacttcaaa gcgtttccga aaacgagcgc
     1741 ttccgaaaat gcaacgcgag ctgcgcacat acagctcact gttcacgtcg cacctatatc
     1801 tgcgtgttgc ctgtatatat atatacatga gaagaacggc atagtgcgtg tttatgctta
     1861 aatgcgtact tatatgcgtc tatttatgta ggatgaaagg tagtctagta cctcctgtga
     1921 tattatccca ttccatgcgg ggtatcgtat gcttccttca gcactaccct ttagctgttc
     1981 tatatgctgc cactcctcaa ttggattagt ctcatccttc aatgctatca tttcctttga
     2041 tattggatca tatgcatagt accgagaaac tagtgcgaag tagtgatcag gtattgctgt
     2101 tatctgatga gtatacgttg tcctggccac ggcagaagca cgcttatcgc tccaatttcc
     2161 cacaacatta gtcaactccg ttaggccctt cattgaaaga aatgaggtca tcaaatgtct
     2221 tccaatgtga gattttgggc cattttttat agcaaagatt gaataaggcg catttttctt
     2281 caaagcttta ttgtacgatc tgactaagtt atcttttaat aattggtatt cctgtttatt
     2341 gcttgaagaa ttgccggtcc tatttactcg ttttaggact ggttcagaat tcgtaatcat
     2401 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     2461 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     2521 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     2581 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     2641 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     2701 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     2761 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     2821 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     2881 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     2941 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3001 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3061 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3121 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3181 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3241 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3301 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3361 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3421 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     3481 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     3541 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     3601 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     3661 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     3721 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     3781 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     3841 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     3901 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     3961 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4021 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4081 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4141 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     4201 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4261 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     4321 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     4381 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     4441 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     4501 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     4561 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     4621 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     4681 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     4741 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     4801 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     4861 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     4921 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     4981 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagcttgcat gcagatctcg
     5041 actacgtcgt taaggccgtt tctgacagag taaaattctt gagggaactt tcaccattat
     5101 gggaaatggt tcaagaaggt attgacttaa actccatcaa atggtcaggt cattgagtgt
     5161 tttttatttg ttgtattttt ttttttttag agaaaatcct ccaatatata aattaggaat
     5221 catagtttca tgattttctg ttacacctaa ctttttgtgt ggtgccctcc tccttgtcaa
     5281 tattaatgtt aaagtgcaat tctttttcct tatcacgttg agccattagt atcaatttgc
     5341 ttacctgtat tcctttacat cctccttttt ctccttcttg ataaatgtat gtagattgcg
     5401 tatatagttt cgtctaccct atgaacatat tccattttgt aatttcgtgt cgtttctatt
     5461 atgaatttca tttataaagt ttatgtacaa atatcataaa aaaagagaat ctttttaagc
     5521 aaggattttc ttaacttctt cggcgacagc atcaccgact tcggtggtac tgttggaacc
     5581 acctaaatca ccagttctga tacctgcatc caaaaccttt ttaactgcat cttcaatggc
     5641 cttaccttct tcaggcaagt tcaatgacaa tttcaacatc attgcagcag acaagatagt
     5701 ggcgataggg ttgaccttat tctttggcaa atctggagca gaaccgtggc atggttcgta
     5761 caaaccaaat gcggtgttct tgtctggcaa agaggccaag gacgcagatg gcaacaaacc
     5821 caaggaacct gggataacgg aggcttcatc ggagatgata tcaccaaaca tgttgctggt
     5881 gattataata ccatttaggt gggttgggtt cttaactagg atcatggcgg cagaatcaat
     5941 caattgatgt tgaaccttca atgtagggaa ttcgttcttg atggtttcct ccacagtttt
     6001 tctccataat cttgaagagg ccaaaacatt agctttatcc aaggaccaaa taggcaatgg
     6061 tggctcatgt tgtagggcca tgaaagcggc cattcttgtg attctttgca cttctggaac
     6121 ggtgtattgt tcactatccc aagcgacacc atcaccatcg tcttcctttc tcttaccaaa
     6181 gtaaatacct cccactaatt ctctgacaac aacgaagtca gtacctttag caaattgtgg
     6241 cttgattgga gataagtcta aaagagagtc ggatgcaaag ttacatggtc ttaagttggc
     6301 gtacaattga agttctttac ggatttttag taaaccttgt tcaggtctaa cactaccggt
     6361 accccattta ggaccaccca cagcacctaa caaaacggca tcagccttct tggaggcttc
     6421 cagcgcctca tctggaagtg gaacacctgt agcatcgata gcagcaccac caattaaatg
     6481 attttcgaaa tcgaacttga cattggaacg aacatcagaa atagctttaa gaaccttaat
     6541 ggcttcggct gtgatttctt gaccaacgtg gtcacctggc aaaacgacga tcttcttagg
     6601 ggcagacatt agaatggtat atccttgaaa tatatatata tattgctgaa atgtaaaagg
     6661 taagaaaagt tagaaagtaa gacgattgct aaccacctat tggaaaaaac aataggtcct
     6721 taaataatat tgtcaacttc aagtattgtg atgcaagcat ttagtcatga acgcttctct
     6781 attctatatg aaaagccggt tccggcgctc tcacctttcc tttttctccc aatttttcag
     6841 ttgaaaaagg tatatgcgtc aggcgacctc tgaaattaac aaaaaatttc cagtcatcga
     6901 atttgattct gtgcgatagc gcccctgtgt gttctcgtta tgttgaggaa aaaaataatg
     6961 gttgctaaga gattcgaact cttgcatctt acgatacctg agtattccca cagttaactg
     7021 cggtcaagat atttcttgaa tcaggcgcct tagaccgctc ggccaaacaa ccaattactt
     7081 gttgagaaat agagtataat tatcctataa atataacgtt tttgaacaca catgaacaag
     7141 gaagtacagg acaattgatt ttgaagagaa tgtggatttt gatgtaattg ttgggattcc
     7201 atttttaata aggcaataat attaggtatg tagatatact agaagttctc ctcgactaga
     7261 gcacattctg cggcctctgt gctctgcaag ccgcaaactt tcaccaatgg accagaacta
     7321 cctgtgaaat taataacaga catactccaa gctgcctttg tgtgcttaat cacgtatact
     7381 cacgtgctca atagtcacca atgccctccc tcttggccct ctccttttct tttttcgacc
     7441 gaatttcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg
     7501 aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt
     7561 cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag
     7621 tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct
     7681 gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgatcc ggagctttaa
     7741 cgaacgcaga attttcgagt tattaaactt aaaatacgct gaacccgaac atagaaatat
     7801 cgaatgggaa aaaaaaactg cataaaggca ttaaaagagg agcgaatttt tttttaataa
     7861 aaatcttaat aatcattaaa agataaataa tagtctatat atacgtatat aaataaaaaa
     7921 tattcaaaaa ataaaataaa ctattatttt agcgtaaagg atggggaaag agaaaagaaa
     7981 aaaattgatc tatcgatttc aattcaattc aatttatttc ttttcggata agaaagcaac
     8041 acctggcaat tccttacctt ccaataattc caaagaagca ccaccaccag tagagacatg
     8101 ggagatcccg gatcattact tatctaggtc gaccagcttg ggctgcaggt catacctata
     8161 aattaacaaa gtatctaaac aaaatacata agtgtactca aactgagtag aatcgtcgat
     8221 taaacttcct tctcctttta aaaattaaaa acagcaaata gttaaatgaa tatattaaag
     8281 actattcgtt ttatttccca gagcagcatg atttcttggt ttcttcagac ttgttaccgc
     8341 aggggcattt gtcgtcgctg ttacaccccg ttgggcagct acatgatttt tggcattgtt
     8401 cattattttt gcagctacca cattggcatt ggcactcatg accttcattt tggaagttaa
     8461 ttaattcgct gaacccggta ccaacctcta aacatataat atagcaacaa aaagaatgaa
     8521 gcaattgttg tttcgatgtt agtacatgag acacagtagc aaataaaaga agaatatcac
     8581 tatcacttat cacgaaaata tttcagtttt tttttttcca aatttcaagc ccctatttat
     8641 tccaataata tcgtggttat tacagatcag tcaatatagg aggttatggg agagtgaaaa
     8701 atagtaaaaa aaggtaaaag agaaatctct cgagcaattg ggaccgtgca attcttctta
     8761 cagttaaatg ggatggtgca agcgctagaa cataccagaa tccattgtta attcagtaaa
     8821 ttttcgatct tgggaagaaa aaagcagtaa gcgtgaaaaa tctaaaaggg gcgagctcgc
     8881 cggggatccg tcgaaactaa gttcttggtg ttttaaaact aaaaaaaaga ctaactataa
     8941 aagtagaatt taagaagttt aagaaataga tttacagaat tacaatcaat acctaccgtc
     9001 tttatatact tattagtcaa gtaggggaat aatttcaggg aactggtttc aacctttttt
     9061 ttcagctttt tccaaatcag agagagcaga aggtaataga aggtgtaaga aaatgagata
     9121 gatacatgcg tgggtcaatt gccttgtgtc atcatttact ccaggcaggt tgcatcactc
     9181 cattgaggtt gtgcccgttt tttgcctgtt tgtgcccctg ttctctgtag ttgcgctaag
     9241 agaatggacc tatgaactga tggttggtga agaaaacaat attttggtgc tgggattctt
     9301 tttttttctg gatgccagct taaaaagcgg gctccattat atttagtgga tgccaggaat
     9361 aaactgttca cccagacacc tacgatgtta tatattctgt gtaacccgcc ccctattttg
     9421 ggcatgtacg ggttacagca gaattaaaag gctaattttt tgactaaata aagttaggaa
     9481 aatcactact attaattatt tacgtattct ttgaaatg
//
