LOCUS       sequence-433688-        6123 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multi-copy integration of heterologous genes (AeBlue and RelB) 
            through co-transformation with ToxAmp (toxin-antitoxin-driven gene 
            amplification) modules.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6123)
  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 6123)
  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: pToxAmp12
FEATURES             Location/Qualifiers
     source          1..6123
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1448..1735
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     1605..1628
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     primer_bind     complement(3270..3286)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3270..3286)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3283..3305)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3294..3310
                     /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(3318..3324,3325..3342,3343..3348))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3363..3384
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3501..3518)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3672..4260)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3752..3771)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4431..5222,5223..5291))
                     /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     5054..5073
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5292..5396)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5464..5482
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5520..5542)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5642..5661
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5855..5877
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5869..5886
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5870..5886
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gcaatcaatt ctatctatac tttaaaccgc ggaccgtatt ttgctcgttc cagcctttcc
       61 acgtttttgt tatctaagca acttggcaca tttccctact atactacaaa ccgatacgta
      121 aatacttccc taaatagcat atgaattatt cagtaatttt taaggatcga aactgcacct
      181 caactattcg ttactgtggt tatgttctca tgtattgatg caaatcatgg gatatttgct
      241 caagacgacg gtaaaatgag caaaaatggc acgatcctga aaagagcact tttcaagatt
      301 cgggctacaa aatgcaacat aaaaaatgtt gtattgtcat ctcgagaggg tcttgtatgt
      361 tttattcctc ttatgattag ttcacattag taaaacagat acgcagtgtg ctcttaataa
      421 acaactactc catagcttta tttgcataac aaaactttta agcacaaact taaacaggtg
      481 gagtaatagt tcggcggcga ctcaaattac atttgttgga agaatcgaat agaaaataaa
      541 aaaaagtgta ttatatttga cattcaaaat gggtagcatt aacctgcgta ttgacgatga
      601 acttaaagcg cgttcttacg ccgcgcttga aaaaatgggt gtaactcctt ctgaagcgct
      661 tcgtctcatg ctcgagtata tcgctgacaa tgaacgcttg ccgttcaaac agacactcct
      721 gagtgatgaa gatgctgaac ttgtggagat agtgaaagaa cggcttcgta atcctaagcc
      781 agtacgtgtg acgctggatg aactctgacc tgcagggcga tttaatctct aattattagt
      841 taaagtttta taagcatttt tatgtaacga aaaataaatt ggttcatatt attactgcac
      901 tgtcacttac catggaaaga ccagacaaga agttgccgac acgacagtct gttgaattgg
      961 cttaagtctg ggtccgcttc tttacaaatt tgaagaattt ctcttaaacg atatgtatat
     1021 tcttttcgtt ggaaaagatg tcttccaaaa aaaaaaaccg atgaattagt ggaaccaagg
     1081 aaaaaaaaag aggtatcctt gattaaggaa cagagctcta tatatttaat aaagagtatc
     1141 atctttcaaa gccttttaat tctgctgtaa cccgtacatg cccaaaatag ggggcgggtt
     1201 acacagaata tataacatcg taggtgtctg ggtgaacagt ttattcctgg catccactaa
     1261 atataatgga gcccgctttt taagctggca tccagaaaaa aaaagaatcc cagcaccaaa
     1321 atattgtttt cttcaccaac catcagttca taggtccatt ctcttagcgc aactacagag
     1381 aacaggggca caaacaggca aaaaacgggc acaacctcaa tggagtgatg caacctgcct
     1441 ggagtaaatg atgacacaag gcaattgacc cacgcatgta tctatctcat tttcttacac
     1501 cttctattac cttctgctct ctctgatttg gaaaaagctg aaaaaaaagg ttgaaaccag
     1561 ttccctgaaa ttattcccct acttgactaa taagtatata aagacggtag gtattgattg
     1621 taattctgta aatctatttc ttaaacttct taaattctac ttttatagtt agtctttttt
     1681 ttagttttaa aacaccaaga acttagtttc gaataaacac acataaacaa acaaaatggc
     1741 ttctttggtt aagaaagata tgtgtatcaa gatgactatg gaaggtactg ttaacggtca
     1801 tcactttaag tgtgttggtg aaggtgaagg taaaccattt gaaggtactc aagttgaaaa
     1861 gattagaatt actgaaggtg gtccattgcc atttgcttat gatattttgg ctccatgttg
     1921 tatgtatggt tctaagactt tcattaagca cgtttctggt atcccagact acttcaaaga
     1981 atcttttcca gaaggtttta cctgggaaag aactcaaatc tttgaagatg gtggttactt
     2041 gactattcac caagatactt cattgcaagg taacaacttt atcttcaaag ttaacgttat
     2101 cggtgctaat tttccagcta atggtccagt tatgcaaaag aaaactgctg gttgggaacc
     2161 atgtgttgaa atgttgtatc caagagatgg tgttttgtgc ggtcaatctt tgatggcttt
     2221 gaaatgtact gatggtaatc acttgacctc tcatttgaga actacttaca gatccagaaa
     2281 accatctaac gctgttaaca tgccagaatt tcatttcggt gaccacagaa ttgagatttt
     2341 gaaagctgaa caaggtaagt tctacgaaca atacgaatct gctgttgcta gatattgtga
     2401 agctgctcca tcaaaattgg gtcatcatta atatataact gtctagaaat aaattttttc
     2461 aaagcggccg cattcccaga agattaggga ggtaattaat taataggcag atttctagtt
     2521 cttatatatt actatagata aggaaattat acgcttgtat gcagagataa tttaagtttt
     2581 gctttgttcg ccaccattct agttatgacc tttatctatc attctatttt ttattggaaa
     2641 ggctttacaa aaaacaaaca gtaaaaattc tttaagcgaa gtgaaatcga caatgaatat
     2701 gaaattggta attatcttac attaggactt ttcaatatct gccggcagga tccacgaaaa
     2761 tgatgtgaat gaatacatga aagattcatg agatctgaca acatggtaga cgtgtgtgtc
     2821 tcatggaaat tgatgcagtt gaagacatgt gcgtcacgaa aaaagaaatc aatcctacac
     2881 agggcttaag ggcaaatgta ttcatgtgtg tcacgaaaag tgatgtaact aaatacacga
     2941 ttaccatgga aattaacgta ccttttttgt gcgtgtattg aaatattatg acatattaca
     3001 gaaagggttc gcaagtcctg tttctatgcc tttctcttag taattcacga aataaaccta
     3061 tggtttacga aatgatccac gaaaatcatg ttattattta catcaacata tcgcgaaaat
     3121 tcatgtcatg tccacattaa catcattgca gagcaacaat tcattttcat agagaaattt
     3181 gctactatca cccactagta ctaccattgg tacctactac tttgaattgt actaccgctg
     3241 ggcgtttaaa cgcatgcggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     3301 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     3361 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3421 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3481 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3541 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3601 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3661 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3721 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3781 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3841 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3901 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3961 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4021 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4081 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     4141 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4201 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4261 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4321 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4381 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4441 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4501 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4561 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4621 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4681 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4741 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4801 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4861 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4921 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4981 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     5041 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     5101 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     5161 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5221 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5281 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5341 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5401 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     5461 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     5521 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     5581 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     5641 gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga
     5701 tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg
     5761 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
     5821 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
     5881 gccagtgcat gcgtttaaac cacgaaaagt tcaccataac ttcgaataaa gtcgcggaaa
     5941 aaagtaaaca gctattgcta ctcaaatgag gtttgcagaa gcttgttgaa gcatgatgaa
     6001 gcgttctaaa cgcactattc atcattaaat atttaaagct cataaaattg tattcaattc
     6061 ctattctaaa tggcttttat ttctattaca actattagct ctaaatccat atcctcataa
     6121 gca
//
