LOCUS       sequence-370766-        6490 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Testing a heat inducible protein degradation tag -DHFR P66L using a 
            green fluorescent protein as the reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6490)
  AUTHORS   Peng B, Bandari NC, Lu Z, Howard CB, Scott C, Trau M, Dumsday G, 
            Vickers CE
  TITLE     Engineering eukaryote-like regulatory circuits to expand artificial 
            control mechanisms for metabolic engineering in Saccharomyces 
            cerevisiae.
  JOURNAL   Commun Biol. 2022 Feb 16;5(1):135. doi: 10.1038/s42003-022-03070-z.
  PUBMED    35173283
REFERENCE   2  (bases 1 to 6490)
  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"; journalName: "Commun 
            Biol."; date: "2022-02-16"; volume: "5(1):135. doi"; pages: " 
            10.1038/s42003-022-03070-z"
COMMENT     Sequence Label: pILGFP8EFA
FEATURES             Location/Qualifiers
     source          1..6490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             31..258
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     CDS             complement(836..853)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             856..1566
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     terminator      1576..1846
                     /gene="S. cerevisae PGK1"
                     /label=PGK1 terminator
                     /note="transcription terminator for 3-phosphoglycerate 
                     kinase gene"
     primer_bind     complement(2111..2127)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2111..2127)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2124..2146)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2135..2151
                     /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(2159..2165,2166..2183,2184..2189))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2204..2225
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2342..2359)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2513..3101)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2593..2612)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3272..4063,4064..4132))
                     /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     3895..3914
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4133..4237)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4305..4323
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4361..4383)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4483..4502
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4696..4718
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4710..4727
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4711..4727
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5841..5874)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        join(6094..6490,1..17)
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
ORIGIN
        1 tagcaatcta atctaagttt taattacaaa atgcagattt tcgtcaagac tttgaccggt
       61 aaaaccataa cattggaagt tgaatcttcc gataccatcg acaacgttaa gtcgaaaatt
      121 caagacaagg aaggtatccc tccagatcaa caaagattga tctttgccgg taagcagcta
      181 gaagacggta gaacgctgtc tgattacaac attcagaagg agtccacctt acatcttgtg
      241 ctaaggctaa gaggtggtag acatggttct ggtatcatgg ttagaccatt gaactgtatc
      301 gttgctgttt ctcaaaacat gggtattgga aagaatggtg acttgccttg gccaccattg
      361 agaaatgagt ttaagtactt ccagagaatg accaccactt cttcagttga aggtaaacaa
      421 aacttggtca tcatgggtag aaagacctgg ttttctattc cagaaaagaa caggttgttg
      481 aaggacagaa tcaacatcgt tttgtccagg gaattgaaag aaccaccaag aggtgctcat
      541 ttcttggcaa aatctttgga tgatgccttg agattgatcg aacaaccaga attggcttcc
      601 aaggttgata tggtttggat tgtcggtggt tcatctgttt atcaagaagc catgaatcaa
      661 ccaggtcatt tgaggttgtt cgttaccaga atcatgcaag agtttgagtc tgatactttc
      721 ttccctgaaa ttgacttggg caagtataag ttgttgccag aatatccagg tgtcttgtct
      781 gaagttcaag aagaaaaggg tatcaagtac aagttcgaag tctacgaaaa gaaggatggt
      841 ggtggtggtg gtggttctaa aggtgaagaa ttattcactg gtgttgtccc aattttggtt
      901 gaattagatg gtgatgttaa tggtcacaaa ttttctgtct ccggtgaagg tgaaggtgat
      961 gctacttacg gtaaattgac cttaaaattt atttgtacta ctggtaaatt gccagttcca
     1021 tggccaacct tagtcactac tttcggttat ggtgttcaat gttttgcgag atacccagat
     1081 catatgaaac aacatgactt tttcaagtct gccatgccag aaggttatgt tcaagaaaga
     1141 actatttttt tcaaagatga cggtaactac aagaccagag ctgaagtcaa gtttgaaggt
     1201 gataccttag ttaatagaat cgaattaaaa ggtattgatt ttaaagaaga tggtaacatt
     1261 ttaggtcaca aattggaata caactataac tctcacaatg tttacatcat ggctgacaaa
     1321 caaaagaatg gtatcaaagt taacttcaaa attagacaca acattgaaga tggttctgtt
     1381 caattagctg accattatca acaaaatact ccaattggtg atggtccagt cttgttacca
     1441 gacaaccatt acttatccac tcaatctgcc ttatccaaag atccaaacga aaagagagac
     1501 cacatggtct tgttagaatt tgttactgct gctggtatta cccatggtat ggatgaattg
     1561 tacaaaagat cttaaattga attgaattga aatcgataga tcaatttttt tcttttctct
     1621 ttccccatcc tttacgctaa aataatagtt tattttattt tttgaatatt ttttatttat
     1681 atacgtatat atagactatt atttatcttt taatgattat taagattttt attaaaaaaa
     1741 aattcgctcc tcttttaatg cctttatgca gttttttttt cccattcgat atttctatgt
     1801 tcgggttcag cgtattttaa gtttaataac tcgaaaattc tgcgttcgtt aaagctttcg
     1861 agaaggatat tatttactag taggactatt tgcaaaggga agggatgcta aggtagaggg
     1921 tgaacgttac agaaaagcag gctgggaagc atatttgaga agatgcggcc agcaaaacta
     1981 aaaaactgta ttataagtaa atgcatgtat actaaactca caaattagag cttcaattta
     2041 attatatcag ttattacccg ggaatctcgg tcgtaatgat ttaaatgcat gcaagcttgg
     2101 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     2161 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     2221 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     2281 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     2341 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     2401 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     2461 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2521 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2581 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2641 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2701 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2761 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2821 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2881 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2941 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3001 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3061 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3121 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     3181 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3241 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3301 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3361 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     3421 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3481 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3541 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3601 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3661 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3721 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3781 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3841 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     3901 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     3961 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     4021 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     4081 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     4141 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     4201 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     4261 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     4321 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     4381 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     4441 cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg
     4501 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     4561 gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     4621 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     4681 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttcatttaaa
     4741 tttttttgat tcggtaatct ccgaacagaa ggaagaacga aggaaggagc acagacttag
     4801 attggtatat atacgcatat gtagtgttga agaaacatga aattgcccag tattcttaac
     4861 ccaactgcac agaacaaaaa cctgcaggaa acgaagataa aacatgtcca caaaatcata
     4921 taccagtaga gctgagactc atgcaagtcc ggttgcatcg aaacttttac gtttaatgga
     4981 tgaaaagaag accaatttgt gtgcttctct tgacgttcgt tcgactgatg agctattgaa
     5041 acttgttgaa acgttgggtc catacatttg ccttttgaaa acacacgttg atatcttgga
     5101 tgatttcagt tatgagggta ctgtcgttcc attgaaagca ttggcagaga aatacaagtt
     5161 cttgatattt gaggacagaa aattcgccga tatcggtaac acagtcaaat tacaatatac
     5221 atcgggcgtt taccgtatcg cagaatggtc tgatatcacc aacgcccacg gggttactgg
     5281 tgctggtatt gttgctggct tgaaacaagg tgcgcaagag gtcaccaaag aaccaagggg
     5341 attattgatg cttgctgaat tgtcttccaa gggttctcta gcacacggtg aatatactaa
     5401 gggtaccgtt gatattgcaa agagtgataa agatttcgtt attgggttca ttgctcagaa
     5461 cgatatggga ggaagagaag aagggtttga ttggctaatc atgaccccag gtgtaggttt
     5521 agacgacaaa ggcgatgcat tgggtcagca gtacagaacc gtcgacgaag ttgtaagtgg
     5581 tggatcagat atcatcattg ttggcagagg acttttcgcc aagggtagag atcctaaggt
     5641 tgaaggtgaa agatacagaa atgctggatg ggaagcgtac caaaagagaa tcagcgctcc
     5701 ccattaatta tacaggaaac ttaatagaac aaatcacata tttaatctaa tagccacctg
     5761 cattggcacg gtgcaacact acttcaactt catcttacaa aaagatcacg tgatctgttg
     5821 tattgggatc tctagaccta ataacttcgt atagcataca ttatacgaag ttatattaag
     5881 ggttgctcga gaaagagctc cagaaagcga ccacccaact ttggctgata atagcgtata
     5941 aacaatgcat actttgtacg ttcaaaatac aatgcagtag atatatttat gcatattaca
     6001 tataatacat atcacatagg aagcaacagg cgcgttggac ttttaatttt cgaggaccgc
     6061 gaatccttac atcacaccca atcccccaca agtgatcccc cacacaccat agcttcaaaa
     6121 tgtttctact ccttttttac tcttccagat tttctcggac tccgcgcatc gccgtaccac
     6181 ttcaaaacac ccaagcacag catactaaat ttcccctctt tcttcctcta gggtgtcgtt
     6241 aattacccgt actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg
     6301 tcgaaaaagg caataaaaat ttttatcacg tttctttttc ttgaaaattt ttttttttga
     6361 tttttttctc tttcgatgac ctcccattga tatttaagtt aataaacggt cttcaatttc
     6421 tcaagtttca gtttcatttt tcttgttcta ttacaacttt ttttacttct tgctcattag
     6481 aaagaaagca
//
