LOCUS       sequence-433686-        6023 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Use together with pJE13HD7 and pJE13HD7 derivatives to integrate 
            RelE expression cassette under the control of the CUP1 promoter at 
            ho locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6023)
  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 6023)
  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: pToxamEXP4CUP1
FEATURES             Location/Qualifiers
     source          1..6023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1284..1300)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1284..1300)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1297..1319)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1308..1324
                     /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(1332..1338,1339..1356,1357..1362))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1377..1398
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1515..1532)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1686..2274)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1766..1785)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2445..3236,3237..3305))
                     /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     3068..3087
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3306..3410)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3478..3496
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3534..3556)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3656..3675
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3869..3891
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3883..3900
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3884..3900
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             3919..4947
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             3919..4947
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      4955..5202
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4971..4989)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        5722..5956
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
ORIGIN
        1 atggcgtatt ttctggattt tgacgagcgg gcactaaagg aatggcgaaa gctgggctcg
       61 acggtacgtg aacagttgaa aaagaagctg gttgaagtac ttgagtcacc ccggattgaa
      121 gcaagtatgt agttccattt ggaagaggga atgaaagaac caagacggtg actttttttt
      181 tagtgttgtg caaccaatat gtcgtgtgta tatcatggta caggagaatg tcaatcagct
      241 aagtgtactc aacatatttc tttgtgtttt gattgcgaac tttgtattac catctcactg
      301 ttgagacggc ttatttgagg taatagctcg agtaaatgta ctcttccatc gcaaactgag
      361 caaaaagaaa gtgtgcatag cctttgtcat acttctcctt tattatacca tgatattcag
      421 aacagtcata ctgtctactc attttacggc tataaaaggt aactttcatt tagattatgg
      481 aaagcactaa ttatcgctgt atcaaatggt tgtagagagc gcaattatga aaaagagtta
      541 ccacgtttct tttgtttcga taaaatgtcc agttgaaaac ctgttttact aacgatttaa
      601 aaattgtatt tcattacaat attttttttg tacagacaag ctccgtggta tgcctgattg
      661 ttacaagatt aagctccggt cttcaggcta tcgccttgta taccaggtta tagacgagaa
      721 agttgtcgtt ttcgtgattt ctgttgggaa aagagaacgc tcggaagtat atagcgaggc
      781 ggtcaaacgc attctctgac ctgcagggat taatataatt atataaaaat attatcttct
      841 tttctttata tctagtgtta tgtaaaataa attgatgact acggaaagct tttttatatt
      901 gtttcttttt cattctgagc cacttaaatt tcgtgaatgt tcttataagg gacggtagat
      961 ttacaagtga tacaacaaaa agcaaggcgc tttttctaat aaaaagaaga aaagcattta
     1021 acaattgaac acctctatat caacgaagaa tattactttg tctctaaatc cttgtaaaat
     1081 gtgtacgatc tctatatggg ttactcagcg gccgcccatt tttagacttt ttcttaacta
     1141 gaatgctgga gtagaaatac gccatctcaa gatacaaaaa gcgttaccgg cactgatttg
     1201 tttcaaccag tatatagatt attattgggt cttgatcaac tttcctcaga catatcagta
     1261 acagtttaaa cggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     1321 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     1381 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     1441 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     1501 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     1561 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     1621 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     1681 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     1741 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     1801 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     1861 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     1921 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     1981 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     2041 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     2101 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     2161 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     2221 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     2281 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     2341 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     2401 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     2461 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     2521 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     2581 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     2641 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     2701 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     2761 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     2821 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     2881 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     2941 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     3001 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     3061 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     3121 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     3181 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     3241 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     3301 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     3361 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     3421 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     3481 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
     3541 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     3601 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
     3661 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta
     3721 aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg
     3781 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     3841 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
     3901 gcatgcgttt aaacaaccat gggtaaaaag cctgaactca ccgcgacgtc tgtcgagaag
     3961 tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa
     4021 tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc
     4081 gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg
     4141 attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc
     4201 cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag
     4261 ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc
     4321 ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg
     4381 attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc
     4441 gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac
     4501 ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg
     4561 gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc
     4621 ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat
     4681 ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa
     4741 ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc
     4801 gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc
     4861 gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc
     4921 agcactcgtc cgagggcaaa ggaataatct cgagtcatgt aattagttat gtcacgctta
     4981 cattcacgcc ctccccccac atccgctcta accgaaaagg aaggagttag acaacctgaa
     5041 gtctaggtcc ctatttattt ttttatagtt atgttagtat taagaacgtt atttatattt
     5101 caaatttttc ttttttttct gtacagacgc gtgtacgcat gtaacattat actgaaaacc
     5161 ttgcttgaga aggttttggg acgctcgaag gctttaacga gctctcgaga acccttaaag
     5221 ttacgctagg gataacaggg taatatagtc tagaatgatt acttatcacg acgcattcgc
     5281 gaaagcgaac cattaccttg atgatgcaga tctcccggtc gtcattactc tacatggacg
     5341 ctttagccag ggctggtatt tctgtttcga agcacgagaa tttctcgaaa ctggagatga
     5401 ggccgcgcgc ttagctggta acgcaccttt tattattgat aaagacagtg gtgaaattca
     5461 ttctctggga acggcaaaac cgctggaaga atatctacag gattacgaaa taaaaaaggc
     5521 taccttcggc ttgccctgag ggcccgggta agccgatccc attaccgaca tttgggcgct
     5581 atacgtgcat atgttcatgt atgtatctgt atttaaaaca cttttgtatt atttttcctc
     5641 atatatgtgt ataggtttat acggatgatt taattattac ttcaccaccc tttatttcag
     5701 gctgatatct tagccttgtt actagttaga aaaagacatt tttgctgtca gtcactgtca
     5761 agagattctt ttgctggcat ttcttctaga agcaaaaaga gcgatgcgtc ttttccgctg
     5821 aaccgttcca gcaaaaaaga ctaccaacgc aatatggatt gtcagaatca tataaaagag
     5881 aagcaaataa ctccttgtct tgtatcaatt gcattataat atcttcttgt tagtgcaata
     5941 tcatatagaa gtcatcgaaa tagatattaa gaaaaacaaa ctgtacaatc aatcaatcaa
     6001 tcatcacata aaactagtgc aaa
//
