LOCUS       pGR595       10016 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Integrates at the CAN1 locus the BadBoy2 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 10016)
  TITLE     Continuous evolution of user-defined genes at 1-million-times the 
            genomic mutation rate
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 10016)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            //gt12365.cn
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pGR595
FEATURES             Location/Qualifiers
     source          1..10016
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(41..60)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        258..924
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     794..817
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     terminator      6033..6198
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(6088..6112)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            6199..7318
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        6199..6542
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             6543..7115
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      7121..7318
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(7856..7872)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7856..7872)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7869..7891)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7880..7896
                     /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(7904..7910,7911..7928,7929..7934))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7949..7970
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8087..8104)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8258..8846)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8338..8357)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9017..9808,9809..9877))
                     /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     9640..9659
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9878..9982)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
       61 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
      121 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
      181 gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tggtgagtga
      241 attccagttc gagtttatca ttatcaatac tgccatttca aagaatacgt aaataattaa
      301 tagtagtgat tttcctaact ttatttagtc aaaaaattag ccttttaatt ctgctgtaac
      361 ccgtacatgc ccaaaatagg gggcgggtta cacagaatat ataacatcgt aggtgtctgg
      421 gtgaacagtt tattcctggc atccactaaa tataatggag cccgcttttt aagctggcat
      481 ccagaaaaaa aaagaatccc agcaccaaaa tattgttttc ttcaccaacc atcagttcat
      541 aggtccattc tcttagcgca actacagaga acaggggcac aaacaggcaa aaaacgggca
      601 caacctcaat ggagtgatgc aacctgcctg gagtaaatga tgacacaagg caattgaccc
      661 acgcatgtat ctatctcatt ttcttacacc ttctattacc ttctgctctc tctgatttgg
      721 aaaaagctga aaaaaaaggt tgaaaccagt tccctgaaat tattccccta cttgactaat
      781 aagtatataa agacggtagg tattgattgt aattctgtaa atctatttct taaacttctt
      841 aaattctact tttatagtta gtcttttttt tagttttaaa acaccaagaa cttagtttcg
      901 aataaacaca cataaacaaa caaaatgaag aacatcaaga agaaccaagt tatgaacttg
      961 ggtccaaact ctaagttgtt gaaggaatac aagtctcaat tgatcgaatt gaacatcgaa
     1021 caattcgaag ctggtatcgg tttgatcttg ggtgacgctt acatccgatc tcgcgacgaa
     1081 ggtaagacct actgtatgca attcgaatgg aagaacaagg cttacatgga ccacgtttgt
     1141 ttgttgtacg accaatgggt tttgtctcca ccacacaaga aggaaagagt taaccacttg
     1201 ggtaacttgg ttatcacctg gggtgctcaa accttcaagc accaagcttt caacaagttg
     1261 gctaacttgt tcatcgttaa caacaagaaa accatcccaa acaacttggt tgaaaactac
     1321 ttgaccccaa tgtctttggc ttactggttc atggacgacg gtggtaagtg ggactacaac
     1381 aagaactcta ccaacaagtc tatcgttttg aacacccaat ctttcacctt cgaagaagtt
     1441 gaatacttgg ttaagggttt gagaaacaag ttccaattga actgttacgt taagatcaac
     1501 aagaacaagc caatcatcta catcgactct atgtcttact tgatcttcta caacttgatc
     1561 aagccatact tgatcccaca aatgatgtac aagttgccaa acaccatctc ttctgaaacc
     1621 ttcttgaagt aaataaagca atcttgatga ggataatgat ttttttttga atatacataa
     1681 atactaccgt ttttctgcta gattttgtga tgacgtaaat aagtacatat tactttttaa
     1741 gccaagacaa gattaagcat taactttacc cttttctttc taagtttcaa tattagttat
     1801 cactgtttaa aagttatggc gagaacgtcg gcggttaaaa tatattaccc tgaacggaat
     1861 tctatatctt taacagattc caaaccctaa agtgtccgaa ttttcaatag ggcgaacttg
     1921 aagaataacc aaggtcaata atatatcttt tagtataacc ctgaaatttg ccctatagaa
     1981 atctagggtt tctgtgtggt ttccgggtga gtcatacggc ttttttgaat ttcttttttt
     2041 gcagttgtct ctatcaatga aaatttcgag gaagacgata aggttaagat aagtagataa
     2101 gagaatgata cgagataaag cacaaattag cagaaagaag agtggttgcg aacagagtaa
     2161 accgaatcag ggaatccctt tttgcaaaaa catcaattat ccttttcttt tttttacgta
     2221 tatatctgga acagaaatat ataagttact attatactta tagttggatc cagtttttaa
     2281 tctgtcgtca atcgaaagtt tatttcagag ttcttcagac ttcttaactc ctgtaaaaac
     2341 aaaaaaaaaa aaaggcatag catttgagaa tgaccttcca cgagctaaat tgaaagggaa
     2401 gaatttatta gttgaactca agaaagaaga ggatgacgtg ggaaatggca tagaatccct
     2461 tactaaatcg aacactaaac tgaactccat gctggcgaac gaaggtaaga tacacaaagc
     2521 tagtttccag aaaagtgtaa aatttaaact acctgataat atagtgactg aagaaaccgt
     2581 ggaacttaaa gaaataaagg acttgctact acaaatgttg agatgacagc gagagattga
     2641 atcaagatta tccaatatcg aacttcaact cacggaaata ccgaaacata agtaatcata
     2701 tcccttctca cattttttac acaggaagta agcaagttat gttatatttc cgacactata
     2761 attaattctt agcagttaaa ggtgctttgt ctatattaca tttacataca gcttgagtga
     2821 tcctgaccgg atatagggtc ctattttctt acgtgaacgg cttttcttct tgttcccgat
     2881 ggccttcatg tgaaaaagca ctcctcggga ggcggaaaaa tatcaaaagt acggggcgaa
     2941 gtttataatg aagatttatc gatataaatt ttggttattt caggagaaca agaaagctct
     3001 ttacactaaa attatcagag aagaagctga tatattagcc ctaaggagta caccaaaaca
     3061 caatggacta caaagacaaa gcattaaacg acttgagaaa cgtatacgca gacttcgact
     3121 ccttgccatt ggacttcaga caaatcttaa ttaaggatag agctacattg ttgcaaaagg
     3181 aagacgttga aaagaaaatt ttggaaagac aagaagatgc caaaaagtac gctgaatact
     3241 tgaagcaatc agaaatccca gaaagaatct ccttacctaa catcaagaga cataagggtg
     3301 tatcaatctc ctttgaagaa acatctgaag atatggtctt ggaaccaaga ccttttatct
     3361 tcgacggttt gaacatcaga tgtttcagaa gagaaaccat attcagtttg aaaaataaga
     3421 ttttgaacat ggttaaggaa tcttcatcct ttaaaaacgt ttcaagacaa agtgtatctt
     3481 ttatgtactt caaaattttt aataagggta aagttattgc aagtacaaag tctgttaaca
     3541 tctatgaaga taagatcgac gaaagattgg aagatttgtg taacaacttc gatgacgtat
     3601 tgaaaaagat tatagacgtc acctatggtt acgaatcctt attcgttagt gaaacttact
     3661 cttacgtaat attctatgct aaatcaatct actttccaca acctagatgc gttaacaact
     3721 ggggtaacaa catcccaaac atcttgacat tcgattcctt taagcttttc accgcaaaca
     3781 aaaacaacgt tagttgtatc aagcaatgtt caagattttt gtggcaaaaa gatttcaaca
     3841 ctttagaaga aatgatagaa tacaagaacg gtaatatttg tatcgtcaca ccacaattgc
     3901 atatcaacga tgttagagac atcaagtcat tcaacgatat cagattgtac tcagaatccc
     3961 ctattaaaac attttccgtt attgataaca ctatcacata tttgttttac ttcaaggaac
     4021 atttgggtgt tatttttaac atcactaagt caagacatga tagaagagta acaaaatttt
     4081 caccattgtc caagttcagt gacgtcaaaa atatcactgt ttgtttcgat atcgaaagtt
     4141 acttcgaccc agaaaaggaa tctaaccaag ttaacatccc tttcatttgt tgcgcttcaa
     4201 tcatctataa taaggttatt ggtaacatcg tcgattttga aggtagagac tgtgtcgcac
     4261 aaatgatcga atacgttgta gatatttgcg gtgaattgaa cataagttct gttgaattga
     4321 tcgcccataa cggtggtggt tatgatttcc actacatctt gtcatccatg tataatccag
     4381 ctgcaattaa aaacatattg atcagaaaca actcttttat ttcttttaat ttcgcacatg
     4441 atggtgtcaa gttctctgtt aaggacagtt actcattttt gtcgtgtagt ttggccaacg
     4501 cttctaaagc atttttaaac gaaggaactt ttaagaaaac tgatttccct catcacgact
     4561 tgaaaactgc cgatgactta tataaggtct acaaggaatg gagttctgtt aacacagaaa
     4621 ttaaccatgt cgttgaaaag gaaaagttgt tgatcacctc tgaacacatc gttaacttca
     4681 ccaagaacga taagagtaag actttgatcg aatggtctaa ggattactgt agaaacgacg
     4741 tattggtctt gtctaaggtt tggttggagt ttaaaaacgc tgtagaagat atttttaact
     4801 gcgaattggt agaccaaacc atgactttgg ctggtttgtc atacaagttg ttccaagcaa
     4861 acatgccatt cgatgtcgaa ttgagacatc ctaataagga agactacttc aacatgagag
     4921 aagcattgat tggtggtaga tgtatctctg ttaacggtat ctacagggat gttttgtgct
     4981 tagacgtaaa atcattatat ccagcttcta tggctttcta tgatcaacct tacggttctt
     5041 ttaagagagt ttcatccaga ccaaaagatg aattgggtat ctattacgtt agagtaacca
     5101 caaacagaaa caacaagtca aactttttcc ctatcagatc ccataataag atcacttaca
     5161 acaacttcga agaatcaact tatattgctt ggtacacaaa cgttgatatt gacattggtt
     5221 tgtcagaagg tcacaacatc gaatacatcc ctttcgattc ctatggtaac attggttact
     5281 catggtccaa aaagggtaaa atttttgaaa agtacatcag ggatgttttg tacaaattga
     5341 agataaaata tgaaaagcaa aataacaagg taaaaagaaa tgtcatcaaa aagatcatga
     5401 actcattgtg gggtaaattc gcccaaaagt gggttaactt cgaatacttc atcaagtcag
     5461 aagatgacat tgatttcgaa tccgaagaag catataaaat ttgggataca gacttcatgt
     5521 tgatcaaaaa gattaaggaa tctatctaca gttctaagcc aatacaaaac ggtgttttta
     5581 ctttaagttg ggccagatat cacatgaagt ctatttggga tgcaggtgcc aaagaaggtg
     5641 ctgaatgtat ctattcagat acagactcca tattcgttca taaggaacac ttcaaaaaga
     5701 atgcaaagtt tatgttgaac ggtttgaaag taccaatcat cggttctgaa gtcggtcaat
     5761 ctgaattaga atgtgaattt gataaattgt tgtgcgccgg taaaaagcaa tacatgggtt
     5821 tctacactta cttccaagat ggtaaacctt gcatcaagga aaagaaaaga ttcaaaggta
     5881 taccaagtaa ctacatcata cctgaattgt atgctcactt gttatctggt gcagataagg
     5941 aagccaaaat tcaatattta aaattcagaa gagaatgggg ttccgtcaag ggttacatag
     6001 aaaataagac tgtcaaagcc acctaactcg aggcgaattt cttatgattt atgattttta
     6061 ttattaaata agttataaaa aaaataagtg tatacaaatt ttaaagtgac tcttaggttt
     6121 taaaacgaaa attcttattc ttgagtaact ctttcctgta ggtcaggttg ctttctcagg
     6181 tatagcatga ggtcgctcga catggaggcc cagaataccc tccttgacag tcttgacgtg
     6241 cgcagctcag gggcatgatg tgactgtcgc ccgtacattt agcccataca tccccatgta
     6301 taatcatttg catccataca ttttgatggc cgcacggcgc gaagcaaaaa ttacggctcc
     6361 tcgctgcaga cctgcgagca gggaaacgct cccctcacag acgcgttgaa ttgtccccac
     6421 gccgcgcccc tgtagagaaa tataaaaggt taggatttgc cactgaggtt cttctttcat
     6481 atacttcctt ttaaaatctt gctaggatac agttctcaca tcacatccga acataaacaa
     6541 ccatgggtac cactcttgac gacacggctt accggtaccg caccagtgtc ccgggggacg
     6601 ccgaggccat cgaggcactg gatgggtcct tcaccaccga caccgtcttc cgcgtcaccg
     6661 ccaccgggga cggcttcacc ctgcgggagg tgccggtgga cccgcccctg accaaggtgt
     6721 tccccgacga cgaatcggac gacgaatcgg acgacgggga ggacggcgac ccggactccc
     6781 ggacgttcgt cgcgtacggg gacgacggcg acctggcggg cttcgtggtc gtctcgtact
     6841 ccggctggaa ccgccggctg accgtcgagg acatcgaggt cgccccggag caccgggggc
     6901 acggggtcgg gcgcgcgttg atggggctcg cgacggagtt cgcccgcgag cggggcgccg
     6961 ggcacctctg gctggaggtc accaacgtca acgcaccggc gatccacgcg taccggcgga
     7021 tggggttcac cctctgcggc ctggacaccg ccctgtacga cggcaccgcc tcggacggcg
     7081 agcaggcgct ctacatgagc atgccctgcc cctaatcagt actgacaata aaaagattct
     7141 tgttttcaag aacttgtcat ttgtatagtt tttttatatt gtagttgttc tattttaatc
     7201 aaatgttagc gtgatttata ttttttttcg cctcgacatc atctgcccag atgcgaagtt
     7261 aagtgcgcag aaagtaatat catgcgtcaa tcgtatgtga atgctggtcg ctatactgat
     7321 atgacgtttt attacctttg atcacatttc cacgccattt cgcattctca ccctcataag
     7381 tcatacaccg aaaagaaagt ttaagggatc aatgagctta ctataatctc agtatattta
     7441 tttttatcga tgattcacca caacaatctt gctcccgaaa agaaagcaga cggagtagaa
     7501 gcatttgaaa ctccttcaga ccttcaagta tatatatata tatatatgta tatgtgtaca
     7561 ttttcacgct aatactaatg tataattaga agataatttt tactcatttt tcgttatctt
     7621 cacgtcaccc gaacctagaa ccaaatgtca ttttcacgat atgtaaatag tgaaataggc
     7681 aaaaacgcca aaaagtagta agcgcaacat acactaaacc attaaagaat atctcgacca
     7741 gaatctaaca gatatacatg ttccgataat gtctgagtta ggtgagtatt ctaaattaga
     7801 aaacaaagag cttagaattc ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     7861 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     7921 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     7981 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     8041 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     8101 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     8161 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     8221 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     8281 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     8341 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     8401 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     8461 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     8521 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     8581 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     8641 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     8701 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     8761 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     8821 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     8881 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     8941 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     9001 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     9061 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     9121 gcttaccatc tggccccagt gctgcaatga taccgcggga cccacgctca ccggctccag
     9181 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     9241 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     9301 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     9361 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     9421 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     9481 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     9541 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     9601 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     9661 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     9721 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     9781 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     9841 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     9901 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     9961 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgac
//