LOCUS       sequence-424625-       10014 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integrates at the CAN1 locus the BadBoy3 TP-DNAP1 variant and the 
            Nourseothricin resistance marker. Includes an I-SceI transient 
            expression cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10014)
  TITLE     Continuous evolution of user-defined genes at 1-million-times the 
            genomic mutation rate
  JOURNAL   Unpublished
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: 
            "Unpublished"
COMMENT     Sequence Label: pGR596
FEATURES             Location/Qualifiers
     source          1..10014
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(35..54)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        252..918
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     788..811
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     terminator      6027..6192
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(6082..6106)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            6193..7312
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        6193..6536
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             6537..7109
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      7115..7312
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(7848..7864)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7848..7864)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7861..7883)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7872..7888
                     /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(7896..7902,7903..7920,7921..7926))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7941..7962
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8079..8096)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8250..8838)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8330..8349)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9009..9800,9801..9869))
                     /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     9632..9651
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9870..9974)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
       61 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
      121 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
      181 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttggtga gtgaattcca
      241 gttcgagttt atcattatca atactgccat ttcaaagaat acgtaaataa ttaatagtag
      301 tgattttcct aactttattt agtcaaaaaa ttagcctttt aattctgctg taacccgtac
      361 atgcccaaaa tagggggcgg gttacacaga atatataaca tcgtaggtgt ctgggtgaac
      421 agtttattcc tggcatccac taaatataat ggagcccgct ttttaagctg gcatccagaa
      481 aaaaaaagaa tcccagcacc aaaatattgt tttcttcacc aaccatcagt tcataggtcc
      541 attctcttag cgcaactaca gagaacaggg gcacaaacag gcaaaaaacg ggcacaacct
      601 caatggagtg atgcaacctg cctggagtaa atgatgacac aaggcaattg acccacgcat
      661 gtatctatct cattttctta caccttctat taccttctgc tctctctgat ttggaaaaag
      721 ctgaaaaaaa aggttgaaac cagttccctg aaattattcc cctacttgac taataagtat
      781 ataaagacgg taggtattga ttgtaattct gtaaatctat ttcttaaact tcttaaattc
      841 tacttttata gttagtcttt tttttagttt taaaacacca agaacttagt ttcgaataaa
      901 cacacataaa caaacaaaat gaagaacatc aagaagaacc aagttatgaa cttgggtcca
      961 aactctaagt tgttgaagga atacaagtct caattgatcg aattgaacat cgaacaattc
     1021 gaagctggta tcggtttgat cttgggtgac gcttacatcc gatctcgcga cgaaggtaag
     1081 acctactgta tgcaattcga atggaagaac aaggcttaca tggaccacgt ttgtttgttg
     1141 tacgaccaat gggttttgtc tccaccacac aagaaggaaa gagttaacca cttgggtaac
     1201 ttggttatca cctggggtgc tcaaaccttc aagcaccaag ctttcaacaa gttggctaac
     1261 ttgttcatcg ttaacaacaa gaaaaccatc ccaaacaact tggttgaaaa ctacttgacc
     1321 ccaatgtctt tggcttactg gttcatggac gacggtggta agtgggacta caacaagaac
     1381 tctaccaaca agtctatcgt tttgaacacc caatctttca ccttcgaaga agttgaatac
     1441 ttggttaagg gtttgagaaa caagttccaa ttgaactgtt acgttaagat caacaagaac
     1501 aagccaatca tctacatcga ctctatgtct tacttgatct tctacaactt gatcaagcca
     1561 tacttgatcc cacaaatgat gtacaagttg ccaaacacca tctcttctga aaccttcttg
     1621 aagtaaataa agcaatcttg atgaggataa tgattttttt ttgaatatac ataaatacta
     1681 ccgtttttct gctagatttt gtgatgacgt aaataagtac atattacttt ttaagccaag
     1741 acaagattaa gcattaactt tacccttttc tttctaagtt tcaatattag ttatcactgt
     1801 ttaaaagtta tggcgagaac gtcggcggtt aaaatatatt accctgaacg gaattctata
     1861 tctttaacag attccaaacc ctaaagtgtc cgaattttca atagggcgaa cttgaagaat
     1921 aaccaaggtc aataatatat cttttagtat aaccctgaaa tttgccctat agaaatctag
     1981 ggtttctgtg tggtttccgg gtgagtcata cggctttttt gaatttcttt ttttgcagtt
     2041 gtctctatca atgaaaattt cgaggaagac gataaggtta agataagtag ataagagaat
     2101 gatacgagat aaagcacaaa ttagcagaaa gaagagtggt tgcgaacaga gtaaaccgaa
     2161 tcagggaatc cctttttgca aaaacatcaa ttatcctttt ctttttttta cgtatatatc
     2221 tggaacagaa atatataagt tactattata cttatagttg gatccagttt ttaatctgtc
     2281 gtcaatcgaa agtttatttc agagttcttc agacttctta actcctgtaa aaacaaaaaa
     2341 aaaaaaaggc atagcatttg agaatgacct tccacgagct aaattgaaag ggaagaattt
     2401 attagttgaa ctcaagaaag aagaggatga cgtgggaaat ggcatagaat cccttactaa
     2461 atcgaacact aaactgaact ccatgctggc gaacgaaggt aagatacaca aagctagttt
     2521 ccagaaaagt gtaaaattta aactacctga taatatagtg actgaagaaa ccgtggaact
     2581 taaagaaata aaggacttgc tactacaaat gttgagatga cagcgagaga ttgaatcaag
     2641 attatccaat atcgaacttc aactcacgga aataccgaaa cataagtaat catatccctt
     2701 ctcacatttt ttacacagga agtaagcaag ttatgttata tttccgacac tataattaat
     2761 tcttagcagt taaaggtgct ttgtctatat tacatttaca tacagcttga gtgatcctga
     2821 ccggatatag ggtcctattt tcttacgtga acggcttttc ttcttgttcc cgatggcctt
     2881 catgtgaaaa agcactcctc gggaggcgga aaaatatcaa aagtacgggg cgaagtttat
     2941 aatgaagatt tatcgatata aattttggtt atttcaggag aacaagaaag ctctttacac
     3001 taaaattatc agagaagaag ctgatatatt agccctaagg agtacaccaa aacacaatgg
     3061 actacaaaga caaagcatta aacgacttga gaaacgtata cgcagacttc gactccttgc
     3121 cattggactt cagacaaatc ttaattaagg atagagctac attgttgcaa aaggaagacg
     3181 ttgaaaagaa aattttggaa agacaagaag atgccaaaaa gtacgctgaa tacttgaagc
     3241 aatcagaaat cccagaaaga atctccttac ctaacatcaa gagacataag ggtgtatcaa
     3301 tctcctttga agaaacatct gaagatatgg tcttggaacc aagacctttt atcttcgacg
     3361 gtttgaacat cagatgtttc agaagagaaa ccatattcag tttgaaaaat aagattttga
     3421 acatggttaa ggaatcttca tcctttaaaa acgtttcaag acaaagtgta tcttttatgt
     3481 acttcaaaat ttttaataag ggtaaagtta ttgcaagtac aaagtctgtt aacatctatg
     3541 aagataagat cgacgaaaga ttggaagatt tgtgtaacaa cttcgatgac gtattgaaaa
     3601 agattataga cgtcacctat ggttacgaat ccttattcgt tagtgaaact tactcttacg
     3661 taatattcta tgctaaatca atctactttc cacaacctag atgcgttaac aactggggta
     3721 acaacatccc aaacatcttg acattcgatt cctttaagct tttcaccgca aacaaaaaca
     3781 acgttagttg tatcaagcaa tgttcaagat ttttgtggca aaaagatttc aacactttag
     3841 aagaaatgat agaatacaag aacggtaata tttgtatcgt cacaccacaa ttgcatatca
     3901 gcgatgttag agacatcaag tcattcaacg atatcagatt gtactcagaa tcccctatta
     3961 aaacattttc cgttattgat aacactatca catatttgtt ttacttcaag gaacatttgg
     4021 gtgttatttt taacttcact aagtcaagac atgatagaag agtaacaaaa ttttcaccat
     4081 tgtccaagtt cagtgacgtc aaaaatatca ctgtttgttt cgatatcgaa agttacttcg
     4141 acccagaaaa ggaatctaac caagttaaca tccctttcat ttgttgcgct tcaatcatct
     4201 ataataaggt tattggtaac atcgtcgatt ttgaaggtag agactgtgtc gcacaaatga
     4261 tcgaatacgt tgtagatatt tgcggtgaat tgaacataag ttctgttgaa ttgatcgccc
     4321 ataacggtgg tggttatgat ttccactaca tcttgtcatc catgtataat ccagctgcaa
     4381 ttaaaaacat attgatcaga aacaactctt ttatttcttt taatttcgca catgatggtg
     4441 tcaagttctc tgttaaggac agttactcat ttttgtcgtg tagtttggcc aacgcttcta
     4501 aagcattttt aaacgaagga acttttaaga aaactgattt ccctcatcac gacttgaaaa
     4561 ctgccgatga cttatataag gtctacaagg aatggagttc tgttaacaca gaaattaacc
     4621 atgtcgttga aaaggaaaag ttgttgatca cctctgaaca catcgttaac ttcaccaaga
     4681 acgataagag taagactttg atcgaatggt ctaaggatta ctgtagaaac gacgtattgg
     4741 tcttgtctaa ggtttggttg gagtttaaaa acgctgtaga agatattttt aactgcgaat
     4801 tggtagacca aaccatgact ttggctggtt tgtcatacaa gttgttccaa gcaaacatgc
     4861 cattcgatgt cgaattgaga catcctaata aggaagacta cttcaacatg agagaagcat
     4921 tgattggtgg tagatgtatc tctgttaacg gtatctacag ggatgttttg tgcttagacg
     4981 taaaatcatt atatccagct tctatggctt tctatgatca accttacggt tcttttaaga
     5041 gagtttcatc cagaccaaaa gatgaattgg gtatctatta cgttagagta accacaaaca
     5101 gaaacaacaa gtcaaacttt ttccctatca gatcccataa taagatcact tacaacaact
     5161 tcgaagaatc aacttatatt gcttggtaca caaacgttga tattgacatt ggtttgtcag
     5221 aaggtcacaa catcgaatac atccctttcg attcctatgg taacattggt tactcatggt
     5281 ccaaaaaggg taaaattttt gaaaagtaca tcagggatgt tttgtacaaa ttgaagataa
     5341 aatatgaaaa gcaaaataac aaggtaaaaa gaaatgtcat caaaaagatc atgaactcat
     5401 tgtggggtaa attcgcccaa aagtgggtta acttcgaata cttcatcaag tcagaagatg
     5461 acattgattt cgaatccgaa gaagcatata aaatttggga tacagacttc atgttgatca
     5521 aaaagattaa ggaatctatc tacagttcta agccaataca aaacggtgtt tttactttaa
     5581 gttgggccag atatcacatg aagtctattt gggatgcagg tgccaaagaa ggtgctgaat
     5641 gtatctattc agatacagac tccatattcg ttcataagga acacttcaaa aagaatgcaa
     5701 agtttatgtt gaacggtttg aaagtaccaa tcatcggttc tgaagtcggt caatctgaat
     5761 tagaatgtga atttgataaa ttgttgtgcg ccggtaaaaa gcaatacatg ggtttctaca
     5821 cttacttcca agatggtaaa ccttgcatca aggaaaagaa aagattcaaa ggtataccaa
     5881 gtaactacat catacctgaa ttgtatgctc acttgttatc tggtgcagat aaggaagcca
     5941 aaattcaata tttaaaattc agaagagaat ggggttccgt caagggttac atagaaaata
     6001 agactgtcaa agccacctaa ctcgaggcga atttcttatg atttatgatt tttattatta
     6061 aataagttat aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac
     6121 gaaaattctt attcttgagt aactctttcc tgtaggtcag gttgctttct caggtatagc
     6181 atgaggtcgc tcgacatgga ggcccagaat accctccttg acagtcttga cgtgcgcagc
     6241 tcaggggcat gatgtgactg tcgcccgtac atttagccca tacatcccca tgtataatca
     6301 tttgcatcca tacattttga tggccgcacg gcgcgaagca aaaattacgg ctcctcgctg
     6361 cagacctgcg agcagggaaa cgctcccctc acagacgcgt tgaattgtcc ccacgccgcg
     6421 cccctgtaga gaaatataaa aggttaggat ttgccactga ggttcttctt tcatatactt
     6481 ccttttaaaa tcttgctagg atacagttct cacatcacat ccgaacataa acaaccatgg
     6541 gtaccactct tgacgacacg gcttaccggt accgcaccag tgtcccgggg gacgccgagg
     6601 ccatcgaggc actggatggg tccttcacca ccgacaccgt cttccgcgtc accgccaccg
     6661 gggacggctt caccctgcgg gaggtgccgg tggacccgcc cctgaccaag gtgttccccg
     6721 acgacgaatc ggacgacgaa tcggacgacg gggaggacgg cgacccggac tcccggacgt
     6781 tcgtcgcgta cggggacgac ggcgacctgg cgggcttcgt ggtcgtctcg tactccggct
     6841 ggaaccgccg gctgaccgtc gaggacatcg aggtcgcccc ggagcaccgg gggcacgggg
     6901 tcgggcgcgc gttgatgggg ctcgcgacgg agttcgcccg cgagcggggc gccgggcacc
     6961 tctggctgga ggtcaccaac gtcaacgcac cggcgatcca cgcgtaccgg cggatggggt
     7021 tcaccctctg cggcctggac accgccctgt acgacggcac cgcctcggac ggcgagcagg
     7081 cgctctacat gagcatgccc tgcccctaat cagtactgac aataaaaaga ttcttgtttt
     7141 caagaacttg tcatttgtat agttttttta tattgtagtt gttctatttt aatcaaatgt
     7201 tagcgtgatt tatatttttt ttcgcctcga catcatctgc ccagatgcga agttaagtgc
     7261 gcagaaagta atatcatgcg tcaatcgtat gtgaatgctg gtcgctatac tgatatgacg
     7321 ttttattacc tttgatcaca tttccacgcc atttcgcatt ctcaccctca taagtcatac
     7381 accgaaaaga aagtttaagg gatcaatgag cttactataa tctcagtata tttattttta
     7441 tcgatgattc accacaacaa tcttgctccc gaaaagaaag cagacggagt agaagcattt
     7501 gaaactcctt cagaccttca agtatatata tatatatatg tatatgtgta cattttcacg
     7561 ctaatactaa tgtataatta gaagataatt tttactcatt tttcgttatc ttcacgtcac
     7621 ccgaacctag aaccaaatgt cattttcacg atatgtaaat agtgaaatag gcaaaaacgc
     7681 caaaaagtag taagcgcaac atacactaaa ccattaaaga atatctcgac cagaatctaa
     7741 cagatataca tgttccgata atgtctgagt taggtgagta ttctaaatta gaaaacaaag
     7801 agcttagaat tcccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     7861 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     7921 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     7981 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     8041 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     8101 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     8161 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     8221 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     8281 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     8341 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     8401 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     8461 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     8521 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     8581 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     8641 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     8701 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     8761 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     8821 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     8881 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     8941 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     9001 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     9061 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     9121 tctggcccca gtgctgcaat gataccgcgg gacccacgct caccggctcc agatttatca
     9181 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     9241 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     9301 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     9361 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     9421 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     9481 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     9541 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     9601 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     9661 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     9721 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     9781 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     9841 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     9901 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     9961 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcg
//
