LOCUS       sequence-433687-        6387 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Amplifying the gene copy number of heterologous genes (yEGFP and 
            RelB) through ToxAmp (toxin-antitoxin-driven gene amplification) 
            mechanism.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6387)
  AUTHORS   Evans S, Lu Z, McDonnell L, Anderson W, Peralta F, Watkins T , Ahmed
            H, Luna-Flores CH, Loan T, Navone L, Trau M, Scott CH, Speight R, 
            Vickers CE, Peng B
  TITLE     The integration of tandem gene repeats via a bacterial type-II 
            toxin-antitoxin-mediated gene amplification (ToxAmp) system and 
            stability visualisation in Saccharomyces cerevisiae
  JOURNAL   bioRxiv 2024.01.30.578080
REFERENCE   2  (bases 1 to 6387)
  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: "bioRxiv 
            2024.01.30.578080"
COMMENT     Sequence Label: pToxAmp1
FEATURES             Location/Qualifiers
     source          1..6387
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(19..35)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(19..35)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(32..54)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    43..59
                     /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(67..73,74..91,92..97))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    112..133
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(250..267)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(421..1009)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(501..520)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1180..1971,1972..2040))
                     /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     1803..1822
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2041..2145)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2213..2231
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2269..2291)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2391..2410
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2604..2626
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2618..2635
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2619..2635
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        4237..4650
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             4673..5383
                     /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"
ORIGIN
        1 gcatgcggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
       61 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
      121 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
      181 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
      241 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
      301 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
      361 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
      421 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
      481 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      541 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      601 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      661 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      721 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      781 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      841 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      901 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      961 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     1021 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     1081 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     1141 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     1201 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     1261 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     1321 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     1381 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1441 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1501 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1561 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1621 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1681 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1741 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     1801 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1861 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1921 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     1981 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     2041 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     2101 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     2161 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     2221 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     2281 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     2341 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     2401 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
     2461 aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     2521 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     2581 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgcatg
     2641 cgtttaaacc acgaaaagtt caccataact tcgaataaag tcgcggaaaa aagtaaacag
     2701 ctattgctac tcaaatgagg tttgcagaag cttgttgaag catgatgaag cgttctaaac
     2761 gcactattca tcattaaata tttaaagctc ataaaattgt attcaattcc tattctaaat
     2821 ggcttttatt tctattacaa ctattagctc taaatccata tcctcataag cagcaatcaa
     2881 ttctatctat actttaaacc gcgggccttg cggaggataa ccaccaacga actcaaatac
     2941 ctaccgcgta ccttcctacg gttatgtact tttgcactgc tattgctttg agatgctatg
     3001 agaactgccg tcaaaattgg gtggcaaaga gggatagctt cactgccaat cactacactc
     3061 tgttctgaag gaggggaatt actgttgtct cataaaaaaa cacctttgaa acactttcac
     3121 tcatagagaa gccaagaact ctgatgagtt ctttatttgt tgccgctaac tttatcttat
     3181 cttctccatt gtaataatta ctattctttt tacttgcctg ttttccatcc caatgaagat
     3241 ccgtacatct tagacaccct tttctttttt tctttttttt tttttttttt tttttggctt
     3301 ccgaagtgcc gacaactaaa gaattatcgc attttttttg aatacatcat actatacgat
     3361 agaacgcatt gaaacttttc ccatctcaaa atccagggac aatagtatgg gatcgatatt
     3421 attatcaaga cataagtatt gtgatgcgca cgttgaaatt cactattttt tttttttata
     3481 tctgtttttc aatacagtga acacgataac tgaaagaaac atctagaatg ggtagcatta
     3541 acctgcgtat tgacgatgaa cttaaagcgc gttcttacgc cgcgcttgaa aaaatgggtg
     3601 taactccttc tgaagcgctt cgtctcatgc tcgagtatat cgctgacaat gaacgcttgc
     3661 cgttcaaaca gacactcctg agtgatgaag atgctgaact tgtggagata gtgaaagaac
     3721 ggcttcgtaa tcctaagcca gtacgtgtga cgctggatga actctgacct gcagggcgat
     3781 ttaatctcta attattagtt aaagttttat aagcattttt atgtaacgaa aaataaattg
     3841 gttcatatta ttactgcact gtcacttacc atggaaagac cagacaagaa gttgccgaca
     3901 cgacagtctg ttgaattggc ttaagtctgg gtccgcttct ttacaaattt gaagaatttc
     3961 tcttaaacga tatgtatatt cttttcgttg gaaaagatgt cttccaaaaa aaaaaaccga
     4021 tgaattagtg gaaccaagga aaaaaaaaga ggtatccttg attaaggaac agagctccgt
     4081 ataaacaatg catactttgt acgttcaaaa tacaatgcag tagatatatt tatgcatatt
     4141 acatataata catatcacat aggaagcaac aggcgcgttg gacttttaat tttcgaggac
     4201 cgcgaatcct tacatcacac ccaatccccc acaagtgatc ccccacacac catagcttca
     4261 aaatgtttct actccttttt tactcttcca gattttctcg gactccgcgc atcgccgtac
     4321 cacttcaaaa cacccaagca cagcatacta aatttcccct ctttcttcct ctagggtgtc
     4381 gttaattacc cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt ttctttttct
     4441 tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt ttcttgaaaa tttttttttt
     4501 tgattttttt ctctttcgat gacctcccat tgatatttaa gttaataaac ggtcttcaat
     4561 ttctcaagtt tcagtttcat ttttcttgtt ctattacaac tttttttact tcttgctcat
     4621 tagaaagaaa gcatagcaat ctaatctaag ttttaattac aaaatgggat cctctaaagg
     4681 tgaagaatta ttcactggtg ttgtcccaat tttggttgaa ttagatggtg atgttaatgg
     4741 tcacaaattt tctgtctccg gtgaaggtga aggtgatgct acttacggta aattgacctt
     4801 aaaatttatt tgtactactg gtaaattgcc agttccatgg ccaaccttag tcactacttt
     4861 cggttatggt gttcaatgtt ttgcgagata cccagatcat atgaaacaac atgacttttt
     4921 caagtctgcc atgccagaag gttatgttca agaaagaact atttttttca aagatgacgg
     4981 taactacaag accagagctg aagtcaagtt tgaaggtgat accttagtta atagaatcga
     5041 attaaaaggt attgatttta aagaagatgg taacatttta ggtcacaaat tggaatacaa
     5101 ctataactct cacaatgttt acatcatggc tgacaaacaa aagaatggta tcaaagttaa
     5161 cttcaaaatt agacacaaca ttgaagatgg ttctgttcaa ttagctgacc attatcaaca
     5221 aaatactcca attggtgatg gtccagtctt gttaccagac aaccattact tatccactca
     5281 atctgcctta tccaaagatc caaacgaaaa gagagaccac atggtcttgt tagaatttgt
     5341 tactgctgct ggtattaccc atggtatgga tgaattgtac aaaagatctt aaactagtag
     5401 gactatttgc aaagggaagg gatgctaagg tagagggtga acgttacaga aaagcaggct
     5461 gggaagcata tttgagaaga tgcggccagc aaaactaaaa aactgtatta taagtaaatg
     5521 catgtatact aaactcacaa attagagctt caatttaatt atatcagtta ttacccggga
     5581 atctcggtcg taatgatttg cggccgcatt cccagaagat tagggaggta attaattaat
     5641 aggcagattt ctagttctta tatattacta tagataagga aattatacgc ttgtatgcag
     5701 agataattta agttttgctt tgttcgccac cattctagtt atgaccttta tctatcattc
     5761 tattttttat tggaaaggct ttacaaaaaa caaacagtaa aaattcttta agcgaagtga
     5821 aatcgacaat gaatatgaaa ttggtaatta tcttacatta ggacttttca atatctgccg
     5881 gcaggatcca cgaaaatgat gtgaatgaat acatgaaaga ttcatgagat ctgacaacat
     5941 ggtagacgtg tgtgtctcat ggaaattgat gcagttgaag acatgtgcgt cacgaaaaaa
     6001 gaaatcaatc ctacacaggg cttaagggca aatgtattca tgtgtgtcac gaaaagtgat
     6061 gtaactaaat acacgattac catggaaatt aacgtacctt ttttgtgcgt gtattgaaat
     6121 attatgacat attacagaaa gggttcgcaa gtcctgtttc tatgcctttc tcttagtaat
     6181 tcacgaaata aacctatggt ttacgaaatg atccacgaaa atcatgttat tatttacatc
     6241 aacatatcgc gaaaattcat gtcatgtcca cattaacatc attgcagagc aacaattcat
     6301 tttcatagag aaatttgcta ctatcaccca ctagtactac cattggtacc tactactttg
     6361 aattgtacta ccgctgggcg tttaaac
//
