LOCUS       sequence-437420-       10014 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the Badboy2 TP-DNAP1 variant under control of a weak 
            promoter for reducing p1 copy number.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10014)
  AUTHORS   Rix G, Williams RL, Hu VJ, Spinner A, Pisera AO, Marks DS, Liu CC
  TITLE     Continuous evolution of user-defined genes at 1 million times the 
            genomic mutation rate.
  JOURNAL   Science. 2024 Nov 8;386(6722):eadm9073. doi: 
            10.1126/science.adm9073. Epub 2024 Nov 8.
  PUBMED    39509492
REFERENCE   2  (bases 1 to 10014)
  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: "Science."; 
            date: "2024-11-8"; volume: "386(6722):eadm9073. doi"; pages: " 
            10.1126/science.adm9073. Epub 2024 Nov 8"
COMMENT     Sequence Label: pGR721 - SLD3p-TP-DNAP1.Badboy2
FEATURES             Location/Qualifiers
     source          1..10014
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        261..927
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     797..820
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     terminator      6036..6201
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(6091..6115)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            6202..7321
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        6202..6545
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             6546..7118
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      7124..7321
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(7857..7873)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7857..7873)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7870..7892)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7881..7897
                     /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(7905..7911,7912..7929,7930..7935))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7950..7971
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8088..8105)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8259..8847)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8339..8358)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9018..9809,9810..9878))
                     /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     9641..9660
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9879..9983)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttggtgag
      241 tgaattccag ttcgagttta tcattatcaa tactgccatt tcaaagaata cgtaaataat
      301 taatagtagt gattttccta actttattta gtcaaaaaat tagcctttta attctgctgt
      361 aacccgtaca tgcccaaaat agggggcggg ttacacagaa tatataacat cgtaggtgtc
      421 tgggtgaaca gtttattcct ggcatccact aaatataatg gagcccgctt tttaagctgg
      481 catccagaaa aaaaaagaat cccagcacca aaatattgtt ttcttcacca accatcagtt
      541 cataggtcca ttctcttagc gcaactacag agaacagggg cacaaacagg caaaaaacgg
      601 gcacaacctc aatggagtga tgcaacctgc ctggagtaaa tgatgacaca aggcaattga
      661 cccacgcatg tatctatctc attttcttac accttctatt accttctgct ctctctgatt
      721 tggaaaaagc tgaaaaaaaa ggttgaaacc agttccctga aattattccc ctacttgact
      781 aataagtata taaagacggt aggtattgat tgtaattctg taaatctatt tcttaaactt
      841 cttaaattct acttttatag ttagtctttt ttttagtttt aaaacaccaa gaacttagtt
      901 tcgaataaac acacataaac aaacaaaatg aagaacatca agaagaacca agttatgaac
      961 ttgggtccaa actctaagtt gttgaaggaa tacaagtctc aattgatcga attgaacatc
     1021 gaacaattcg aagctggtat cggtttgatc ttgggtgacg cttacatccg atctcgcgac
     1081 gaaggtaaga cctactgtat gcaattcgaa tggaagaaca aggcttacat ggaccacgtt
     1141 tgtttgttgt acgaccaatg ggttttgtct ccaccacaca agaaggaaag agttaaccac
     1201 ttgggtaact tggttatcac ctggggtgct caaaccttca agcaccaagc tttcaacaag
     1261 ttggctaact tgttcatcgt taacaacaag aaaaccatcc caaacaactt ggttgaaaac
     1321 tacttgaccc caatgtcttt ggcttactgg ttcatggacg acggtggtaa gtgggactac
     1381 aacaagaact ctaccaacaa gtctatcgtt ttgaacaccc aatctttcac cttcgaagaa
     1441 gttgaatact tggttaaggg tttgagaaac aagttccaat tgaactgtta cgttaagatc
     1501 aacaagaaca agccaatcat ctacatcgac tctatgtctt acttgatctt ctacaacttg
     1561 atcaagccat acttgatccc acaaatgatg tacaagttgc caaacaccat ctcttctgaa
     1621 accttcttga agtaaataaa gcaatcttga tgaggataat gatttttttt tgaatataca
     1681 taaatactac cgtttttctg ctagattttg tgatgacgta aataagtaca tattactttt
     1741 taagccaaga caagattaag cattaacttt acccttttct ttctaagttt caatattagt
     1801 tatcactgtt taaaagttat ggcgagaacg tcggcggtta aaatatatta ccctgaacgg
     1861 aattctatat ctttaacaga ttccaaaccc taaagtgtcc gaattttcaa tagggcgaac
     1921 ttgaagaata accaaggtca ataatatatc ttttagtata accctgaaat ttgccctata
     1981 gaaatctagg gtttctgtgt ggtttccggg tgagtcatac ggcttttttg aatttctttt
     2041 tttgcagttg tctctatcaa tgaaaatttc gaggaagacg ataaggttaa gataagtaga
     2101 taagagaatg atacgagata aagcacaaat tagcagaaag aagagtggtt gcgaacagag
     2161 taaaccgaat cagggaatcc ctttttgcaa aaacatcaat tatccttttc ttttttttac
     2221 gtatatatct ggaacagaaa tatataagtt actattatac ttatagttgg atccagtttt
     2281 taatctgtcg tcaatcgaaa gtttatttca gagttcttca gacttcttaa ctcctgtaaa
     2341 aacaaaaaaa aaaaaaggca tagcatccga gtgagcaatt caggataccg acagcagtag
     2401 tgtggataga ttgtgcaaga ctagtaactg gtaaagggct ccctgataag gccttggagt
     2461 gctccatgat tctcggagtc atatttgccg ttttatcatt aatcagaaac acttatagag
     2521 attacggata cgggtggata ttatatattc cgtctggtgt agcagtcggt gttggtatat
     2581 ttaattctcc cagttttaca attgcaagat tcatcggcgg gtgggcttcg catttttggt
     2641 tgaagaatca taggggtgac ttaaatgcga aaacaaaaat gattgtattc agttcggggt
     2701 tggtcttagg tgaaggtatc tttagcgtaa taaacatgct cttcatctgc ttaaatgtcc
     2761 ctcactatta ggtatataat attttactaa taaagaattt acaaagttta caaattttaa
     2821 attatttact catcacataa ttcgatgtct tagatttccc tgtatatata tttctaggtg
     2881 acaaaaatcc taggcaaaat gttatattat acgcgacctt gaaatatcgg ctatcttggt
     2941 gtacaacatg gtccatttaa gaaaaataaa aaaaagtgat gggcactgtt aaaacaggca
     3001 gaagcctcat tcataactgc tgaagggtta agatcgtata ggtaggaacg atctgacctc
     3061 tcataatgga ctacaaagac aaagcattaa acgacttgag aaacgtatac gcagacttcg
     3121 actccttgcc attggacttc agacaaatct taattaagga tagagctaca ttgttgcaaa
     3181 aggaagacgt tgaaaagaaa attttggaaa gacaagaaga tgccaaaaag tacgctgaat
     3241 acttgaagca atcagaaatc ccagaaagaa tctccttacc taacatcaag agacataagg
     3301 gtgtatcaat ctcctttgaa gaaacatctg aagatatggt cttggaacca agacctttta
     3361 tcttcgacgg tttgaacatc agatgtttca gaagagaaac catattcagt ttgaaaaata
     3421 agattttgaa catggttaag gaatcttcat cctttaaaaa cgtttcaaga caaagtgtat
     3481 cttttatgta cttcaaaatt tttaataagg gtaaagttat tgcaagtaca aagtctgtta
     3541 acatctatga agataagatc gacgaaagat tggaagattt gtgtaacaac ttcgatgacg
     3601 tattgaaaaa gattatagac gtcacctatg gttacgaatc cttattcgtt agtgaaactt
     3661 actcttacgt aatattctat gctaaatcaa tctactttcc acaacctaga tgcgttaaca
     3721 actggggtaa caacatccca aacatcttga cattcgattc ctttaagctt ttcaccgcaa
     3781 acaaaaacaa cgttagttgt atcaagcaat gttcaagatt tttgtggcaa aaagatttca
     3841 acactttaga agaaatgata gaatacaaga acggtaatat ttgtatcgtc acaccacaat
     3901 tgcatatcaa cgatgttaga gacatcaagt cattcaacga tatcagattg tactcagaat
     3961 cccctattaa aacattttcc gttattgata acactatcac atatttgttt tacttcaagg
     4021 aacatttggg tgttattttt aacatcacta agtcaagaca tgatagaaga gtaacaaaat
     4081 tttcaccatt gtccaagttc agtgacgtca aaaatatcac tgtttgtttc gatatcgaaa
     4141 gttacttcga cccagaaaag gaatctaacc aagttaacat ccctttcatt tgttgcgctt
     4201 caatcatcta taataaggtt attggtaaca tcgtcgattt tgaaggtaga gactgtgtcg
     4261 cacaaatgat cgaatacgtt gtagatattt gcggtgaatt gaacataagt tctgttgaat
     4321 tgatcgccca taacggtggt ggttatgatt tccactacat cttgtcatcc atgtataatc
     4381 cagctgcaat taaaaacata ttgatcagaa acaactcttt tatttctttt aatttcgcac
     4441 atgatggtgt caagttctct gttaaggaca gttactcatt tttgtcgtgt agtttggcca
     4501 acgcttctaa agcattttta aacgaaggaa cttttaagaa aactgatttc cctcatcacg
     4561 acttgaaaac tgccgatgac ttatataagg tctacaagga atggagttct gttaacacag
     4621 aaattaacca tgtcgttgaa aaggaaaagt tgttgatcac ctctgaacac atcgttaact
     4681 tcaccaagaa cgataagagt aagactttga tcgaatggtc taaggattac tgtagaaacg
     4741 acgtattggt cttgtctaag gtttggttgg agtttaaaaa cgctgtagaa gatattttta
     4801 actgcgaatt ggtagaccaa accatgactt tggctggttt gtcatacaag ttgttccaag
     4861 caaacatgcc attcgatgtc gaattgagac atcctaataa ggaagactac ttcaacatga
     4921 gagaagcatt gattggtggt agatgtatct ctgttaacgg tatctacagg gatgttttgt
     4981 gcttagacgt aaaatcatta tatccagctt ctatggcttt ctatgatcaa ccttacggtt
     5041 cttttaagag agtttcatcc agaccaaaag atgaattggg tatctattac gttagagtaa
     5101 ccacaaacag aaacaacaag tcaaactttt tccctatcag atcccataat aagatcactt
     5161 acaacaactt cgaagaatca acttatattg cttggtacac aaacgttgat attgacattg
     5221 gtttgtcaga aggtcacaac atcgaataca tccctttcga ttcctatggt aacattggtt
     5281 actcatggtc caaaaagggt aaaatttttg aaaagtacat cagggatgtt ttgtacaaat
     5341 tgaagataaa atatgaaaag caaaataaca aggtaaaaag aaatgtcatc aaaaagatca
     5401 tgaactcatt gtggggtaaa ttcgcccaaa agtgggttaa cttcgaatac ttcatcaagt
     5461 cagaagatga cattgatttc gaatccgaag aagcatataa aatttgggat acagacttca
     5521 tgttgatcaa aaagattaag gaatctatct acagttctaa gccaatacaa aacggtgttt
     5581 ttactttaag ttgggccaga tatcacatga agtctatttg ggatgcaggt gccaaagaag
     5641 gtgctgaatg tatctattca gatacagact ccatattcgt tcataaggaa cacttcaaaa
     5701 agaatgcaaa gtttatgttg aacggtttga aagtaccaat catcggttct gaagtcggtc
     5761 aatctgaatt agaatgtgaa tttgataaat tgttgtgcgc cggtaaaaag caatacatgg
     5821 gtttctacac ttacttccaa gatggtaaac cttgcatcaa ggaaaagaaa agattcaaag
     5881 gtataccaag taactacatc atacctgaat tgtatgctca cttgttatct ggtgcagata
     5941 aggaagccaa aattcaatat ttaaaattca gaagagaatg gggttccgtc aagggttaca
     6001 tagaaaataa gactgtcaaa gccacctaac tcgaggcgaa tttcttatga tttatgattt
     6061 ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg
     6121 ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc
     6181 aggtatagca tgaggtcgct cgacatggag gcccagaata ccctccttga cagtcttgac
     6241 gtgcgcagct caggggcatg atgtgactgt cgcccgtaca tttagcccat acatccccat
     6301 gtataatcat ttgcatccat acattttgat ggccgcacgg cgcgaagcaa aaattacggc
     6361 tcctcgctgc agacctgcga gcagggaaac gctcccctca cagacgcgtt gaattgtccc
     6421 cacgccgcgc ccctgtagag aaatataaaa ggttaggatt tgccactgag gttcttcttt
     6481 catatacttc cttttaaaat cttgctagga tacagttctc acatcacatc cgaacataaa
     6541 caaccatggg taccactctt gacgacacgg cttaccggta ccgcaccagt gtcccggggg
     6601 acgccgaggc catcgaggca ctggatgggt ccttcaccac cgacaccgtc ttccgcgtca
     6661 ccgccaccgg ggacggcttc accctgcggg aggtgccggt ggacccgccc ctgaccaagg
     6721 tgttccccga cgacgaatcg gacgacgaat cggacgacgg ggaggacggc gacccggact
     6781 cccggacgtt cgtcgcgtac ggggacgacg gcgacctggc gggcttcgtg gtcgtctcgt
     6841 actccggctg gaaccgccgg ctgaccgtcg aggacatcga ggtcgccccg gagcaccggg
     6901 ggcacggggt cgggcgcgcg ttgatggggc tcgcgacgga gttcgcccgc gagcggggcg
     6961 ccgggcacct ctggctggag gtcaccaacg tcaacgcacc ggcgatccac gcgtaccggc
     7021 ggatggggtt caccctctgc ggcctggaca ccgccctgta cgacggcacc gcctcggacg
     7081 gcgagcaggc gctctacatg agcatgccct gcccctaatc agtactgaca ataaaaagat
     7141 tcttgttttc aagaacttgt catttgtata gtttttttat attgtagttg ttctatttta
     7201 atcaaatgtt agcgtgattt atattttttt tcgcctcgac atcatctgcc cagatgcgaa
     7261 gttaagtgcg cagaaagtaa tatcatgcgt caatcgtatg tgaatgctgg tcgctatact
     7321 gatatgacgt tttattacct ttgatcacat ttccacgcca tttcgcattc tcaccctcat
     7381 aagtcataca ccgaaaagaa agtttaaggg atcaatgagc ttactataat ctcagtatat
     7441 ttatttttat cgatgattca ccacaacaat cttgctcccg aaaagaaagc agacggagta
     7501 gaagcatttg aaactccttc agaccttcaa gtatatatat atatatatgt atatgtgtac
     7561 attttcacgc taatactaat gtataattag aagataattt ttactcattt ttcgttatct
     7621 tcacgtcacc cgaacctaga accaaatgtc attttcacga tatgtaaata gtgaaatagg
     7681 caaaaacgcc aaaaagtagt aagcgcaaca tacactaaac cattaaagaa tatctcgacc
     7741 agaatctaac agatatacat gttccgataa tgtctgagtt aggtgagtat tctaaattag
     7801 aaaacaaaga gcttagaatt cccgtcgacc tctagctaga gcttggcgta atcatggtca
     7861 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     7921 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     7981 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     8041 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     8101 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     8161 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     8221 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     8281 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     8341 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     8401 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     8461 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     8521 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     8581 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     8641 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     8701 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     8761 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     8821 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     8881 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     8941 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     9001 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     9061 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     9121 ggcttaccat ctggccccag tgctgcaatg ataccgcggg acccacgctc accggctcca
     9181 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     9241 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     9301 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     9361 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     9421 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     9481 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     9541 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     9601 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     9661 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     9721 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     9781 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     9841 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     9901 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     9961 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
