LOCUS       pGR618      10014 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Integrates at the CAN1 locus the BB-Tv 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  JOURNAL   Unpublished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 Apr 14, 2025 from SnapGene Server 8.0.1
            //gt12365.cn
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pGR618
FEATURES             Location/Qualifiers
     source          1..10014
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(28..47)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        245..911
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     781..804
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     terminator      6020..6185
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(6075..6099)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            6186..7305
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        6186..6529
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             6530..7102
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      7108..7305
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     primer_bind     complement(7841..7857)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7841..7857)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7854..7876)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7865..7881
                     /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(7889..7895,7896..7913,7914..7919))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7934..7955
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(8072..8089)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8243..8831)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8323..8342)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9002..9793,9794..9862))
                     /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     9625..9644
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9863..9967)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcccgatccc ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
       61 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
      121 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
      181 tgcgctgctt cgcgatgtac gggccagata tacgcgttgg tgagtgaatt ccagttcgag
      241 tttatcatta tcaatactgc catttcaaag aatacgtaaa taattaatag tagtgatttt
      301 cctaacttta tttagtcaaa aaattagcct tttaattctg ctgtaacccg tacatgccca
      361 aaataggggg cgggttacac agaatatata acatcgtagg tgtctgggtg aacagtttat
      421 tcctggcatc cactaaatat aatggagccc gctttttaag ctggcatcca gaaaaaaaaa
      481 gaatcccagc accaaaatat tgttttcttc accaaccatc agttcatagg tccattctct
      541 tagcgcaact acagagaaca ggggcacaaa caggcaaaaa acgggcacaa cctcaatgga
      601 gtgatgcaac ctgcctggag taaatgatga cacaaggcaa ttgacccacg catgtatcta
      661 tctcattttc ttacaccttc tattaccttc tgctctctct gatttggaaa aagctgaaaa
      721 aaaaggttga aaccagttcc ctgaaattat tcccctactt gactaataag tatataaaga
      781 cggtaggtat tgattgtaat tctgtaaatc tatttcttaa acttcttaaa ttctactttt
      841 atagttagtc ttttttttag ttttaaaaca ccaagaactt agtttcgaat aaacacacat
      901 aaacaaacaa aatgaagaac atcaagaaga accaagttat gaacttgggt ccaaactcta
      961 agttgttgaa ggaatacaag tctcaattga tcgaattgaa catcgaacaa ttcgaagctg
     1021 gtatcggttt gatcttgggt gacgcttaca tccgatctcg cgacgaaggt aagacctact
     1081 gtatgcaatt cgaatggaag aacaaggctt acatggacca cgtttgtttg ttgtacgacc
     1141 aatgggtttt gtctccacca cacaagaagg aaagagttaa ccacttgggt aacttggtta
     1201 tcacctgggg tgctcaaacc ttcaagcacc aagctttcaa caagttggct aacttgttca
     1261 tcgttaacaa caagaaaacc atcccaaaca acttggttga aaactacttg accccaatgt
     1321 ctttggctta ctggttcatg gacgacggtg gtaagtggga ctacaacaag aactctacca
     1381 acaagtctat cgttttgaac acccaatctt tcaccttcga agaagttgaa tacttggtta
     1441 agggtttgag aaacaagttc caattgaact gttacgttaa gatcaacaag aacaagccaa
     1501 tcatctacat cgactctatg tcttacttga tcttctacaa cttgatcaag ccatacttga
     1561 tcccacaaat gatgtacaag ttgccaaaca ccatctcttc tgaaaccttc ttgaagtaaa
     1621 taaagcaatc ttgatgagga taatgatttt tttttgaata tacataaata ctaccgtttt
     1681 tctgctagat tttgtgatga cgtaaataag tacatattac tttttaagcc aagacaagat
     1741 taagcattaa ctttaccctt ttctttctaa gtttcaatat tagttatcac tgtttaaaag
     1801 ttatggcgag aacgtcggcg gttaaaatat attaccctga acggaattct atatctttaa
     1861 cagattccaa accctaaagt gtccgaattt tcaatagggc gaacttgaag aataaccaag
     1921 gtcaataata tatcttttag tataaccctg aaatttgccc tatagaaatc tagggtttct
     1981 gtgtggtttc cgggtgagtc atacggcttt tttgaatttc tttttttgca gttgtctcta
     2041 tcaatgaaaa tttcgaggaa gacgataagg ttaagataag tagataagag aatgatacga
     2101 gataaagcac aaattagcag aaagaagagt ggttgcgaac agagtaaacc gaatcaggga
     2161 atcccttttt gcaaaaacat caattatcct tttctttttt ttacgtatat atctggaaca
     2221 gaaatatata agttactatt atacttatag ttggatccag tttttaatct gtcgtcaatc
     2281 gaaagtttat ttcagagttc ttcagacttc ttaactcctg taaaaacaaa aaaaaaaaaa
     2341 ggcatagcat ttgagaatga ccttccacga gctaaattga aagggaagaa tttattagtt
     2401 gaactcaaga aagaagagga tgacgtggga aatggcatag aatcccttac taaatcgaac
     2461 actaaactga actccatgct ggcgaacgaa ggtaagatac acaaagctag tttccagaaa
     2521 agtgtaaaat ttaaactacc tgataatata gtgactgaag aaaccgtgga acttaaagaa
     2581 ataaaggact tgctactaca aatgttgaga tgacagcgag agattgaatc aagattatcc
     2641 aatatcgaac ttcaactcac ggaaataccg aaacataagt aatcatatcc cttctcacat
     2701 tttttacaca ggaagtaagc aagttatgtt atatttccga cactataatt aattcttagc
     2761 agttaaaggt gctttgtcta tattacattt acatacagct tgagtgatcc tgaccggata
     2821 tagggtccta ttttcttacg tgaacggctt ttcttcttgt tcccgatggc cttcatgtga
     2881 aaaagcactc ctcgggaggc ggaaaaatat caaaagtacg gggcgaagtt tataatgaag
     2941 atttatcgat ataaattttg gttatttcag gagaacaaga aagctcttta cactaaaatt
     3001 atcagagaag aagctgatat attagcccta aggagtacac caaaacacaa tggactacaa
     3061 agacaaagca ttaaacgact tgagaaacgt atacgcagac ttcgactcct tgccattgga
     3121 cttcagacaa atcttaatta aggatagagc tacattgttg caaaaggaag acgttgaaaa
     3181 gaaaattttg gaaagacaag aagatgccaa aaagtacgct gaatacttga agcaatcaga
     3241 aatcccagaa agaatctcct tacctaacat caagagacat aagggtgtat caatctcctt
     3301 tgaagaaaca tctgaagata tggtcttgga accaagacct tttatcttcg acggtttgaa
     3361 catcagatgt ttcagaagag aaaccatatt cagtttgaaa aataagattt tgaacatggt
     3421 taaggaatct tcatccttta aaaacgtttc aagacaaagt gtatctttta tgtacttcaa
     3481 aatttttaat aagggtaaag ttattgcaag tacaaagtct gttaacatct atgaagataa
     3541 gatcgacgaa agattggaag atttgtgtaa caacttcgat gacgtattga aaaagattat
     3601 agacgtcacc tatggttacg aatccttatt cgttagtgaa acttactctt acgtaatatt
     3661 ctatgctaaa tcaatctact ttccacaacc tagatgcgtt aacaactggg gtaacaacat
     3721 cccaaacatc ttgacattcg attcctttaa gcttttcacc gcaaacaaaa acaacgttag
     3781 ttgtatcaag caatgttcaa gatttttgtg gcaaaaagat ttcaacactt tagaagaaat
     3841 gatagaatac aagaacggta atatttgtat cgtcacacca caattgcata tcagcgatgt
     3901 tagagacatc aagtcattca acgatatcag attgtactca gaatccccta ttaaaacatt
     3961 ttccgttatt gataacacta tcacatattt gttttacttc aaggaacatt tgggtgttat
     4021 ttttaacttc actaagtcaa gacatgatag aagagtaaca aaattttcac cattgtccaa
     4081 gttcagtgac gtcaaaaata tcactgtttg tttcgatatc gaaagttact tcgacccaga
     4141 aaaggaatct aaccaagtta acatcccttt catttgttgc gcttcaatca tctataataa
     4201 ggttattggt aacatcgtcg attttgaagg tagagactgt gtcgcacaaa tgatcgaata
     4261 cgttgtagat atttgcggtg aattgaacat aagttctgtt gaattgatcg cccataacgg
     4321 tggtggttat gatttccact acatcttgtc atccatgtat aatccagctg caattaaaaa
     4381 catattgatc agaaacaact cttttatttc ttttaatttc gcacatgatg gtgtcaagtt
     4441 ctctgttaag gacagttact catttttgtc gtgtagtttg gccaacgctt ctaaagcatt
     4501 tttaaacgaa ggaactttta agaaaactga tttccctcat cacgacttga aaactgccga
     4561 tgacttatat aaggtctaca aggaatggag ttctgttaac acagaaatta accatgtcgt
     4621 tgaaaaggaa aagttgttga tcacctctga acacatcgtt aacttcacca agaacgataa
     4681 gagtaagact ttgatcgaat ggtctaagga ttactgtaga aacgacgtat tggtcttgtc
     4741 taaggtttgg ttggagttta aaaacgctgt agaagatatt tttaactgcg aattggtaga
     4801 ccaaaccatg actttggctg gtttgtcata caagttgttc caagcaaaca tgccattcga
     4861 tgtcgaattg agacatccta ataaggaaga ctacttcaac atgagagaag cattgattgg
     4921 tggtagatgt atctctgtta acggtatcta cagggatgtt ttgtgcttag acgtaaaatc
     4981 attatatcca gcttctatgg ctttctatga tcaaccttac ggttctttta agagagtttc
     5041 atccagacca aaagatgaat tgggtatcta ttacgttaga gtaaccacaa acagaaacaa
     5101 caagtcaaac tttttcccta tcagatccca taataagatc acttacaaca acttcgaaga
     5161 atcaacttat attgcttggt acacaaacgt tgatattgac attggtttgt cagaaggtca
     5221 caacatcgaa tacatccctt tcgattccta tggtaacatt ggttactcat ggtccaaaaa
     5281 gggtaaaatt tttgaaaagt acatcaggga tgttttgtac aaattgaaga taaaatatga
     5341 aaagcaaaat aacaaggtaa aaagaaatgt catcaaatct atcatgaact cattgtgggg
     5401 taaattcgcc caaaagtggg ttaacttcga atacttcatc aagtcagaag atgacattga
     5461 tttcgaatcc gaagaagcat ataaaatttg ggatacagac ttcatgttga tcaaaaagat
     5521 taaggaatct atctacagtt ctaagccaat acaaaacggt gtttttactt taagttgggc
     5581 cagatatcac atgaagtcta tttgggatgc aggtgccaaa gaaggtgctg aatgtatcta
     5641 ttcagataca gactccatat tcgttcataa ggaacacttc aaaaagaatg caaagtttat
     5701 gttgaacggt ttgaaagtac caatcatcgg ttctgaagtc ggtcaatctg aattagaatg
     5761 tgaatttgat aaattgttgt gcgccggtaa aaagcaatac atgggtttct acacttactt
     5821 ccaagatggt aaaccttgca tcaaggaaaa gaaaagattc aaaggtatac caagtaacta
     5881 catcatacct gaattgtatg ctcacttgtt atctggtgca gataaggaag ccaaaattca
     5941 atatttaaaa ttcagaagag aatggggttc cgtcaagggt tacatagaaa ataagactgt
     6001 caaagccacc taactcgagg cgaatttctt atgatttatg atttttatta ttaaataagt
     6061 tataaaaaaa ataagtgtat acaaatttta aagtgactct taggttttaa aacgaaaatt
     6121 cttattcttg agtaactctt tcctgtaggt caggttgctt tctcaggtat agcatgaggt
     6181 cgctcgacat ggaggcccag aataccctcc ttgacagtct tgacgtgcgc agctcagggg
     6241 catgatgtga ctgtcgcccg tacatttagc ccatacatcc ccatgtataa tcatttgcat
     6301 ccatacattt tgatggccgc acggcgcgaa gcaaaaatta cggctcctcg ctgcagacct
     6361 gcgagcaggg aaacgctccc ctcacagacg cgttgaattg tccccacgcc gcgcccctgt
     6421 agagaaatat aaaaggttag gatttgccac tgaggttctt ctttcatata cttcctttta
     6481 aaatcttgct aggatacagt tctcacatca catccgaaca taaacaacca tgggtaccac
     6541 tcttgacgac acggcttacc ggtaccgcac cagtgtcccg ggggacgccg aggccatcga
     6601 ggcactggat gggtccttca ccaccgacac cgtcttccgc gtcaccgcca ccggggacgg
     6661 cttcaccctg cgggaggtgc cggtggaccc gcccctgacc aaggtgttcc ccgacgacga
     6721 atcggacgac gaatcggacg acggggagga cggcgacccg gactcccgga cgttcgtcgc
     6781 gtacggggac gacggcgacc tggcgggctt cgtggtcgtc tcgtactccg gctggaaccg
     6841 ccggctgacc gtcgaggaca tcgaggtcgc cccggagcac cgggggcacg gggtcgggcg
     6901 cgcgttgatg gggctcgcga cggagttcgc ccgcgagcgg ggcgccgggc acctctggct
     6961 ggaggtcacc aacgtcaacg caccggcgat ccacgcgtac cggcggatgg ggttcaccct
     7021 ctgcggcctg gacaccgccc tgtacgacgg caccgcctcg gacggcgagc aggcgctcta
     7081 catgagcatg ccctgcccct aatcagtact gacaataaaa agattcttgt tttcaagaac
     7141 ttgtcatttg tatagttttt ttatattgta gttgttctat tttaatcaaa tgttagcgtg
     7201 atttatattt tttttcgcct cgacatcatc tgcccagatg cgaagttaag tgcgcagaaa
     7261 gtaatatcat gcgtcaatcg tatgtgaatg ctggtcgcta tactgatatg acgttttatt
     7321 acctttgatc acatttccac gccatttcgc attctcaccc tcataagtca tacaccgaaa
     7381 agaaagttta agggatcaat gagcttacta taatctcagt atatttattt ttatcgatga
     7441 ttcaccacaa caatcttgct cccgaaaaga aagcagacgg agtagaagca tttgaaactc
     7501 cttcagacct tcaagtatat atatatatat atgtatatgt gtacattttc acgctaatac
     7561 taatgtataa ttagaagata atttttactc atttttcgtt atcttcacgt cacccgaacc
     7621 tagaaccaaa tgtcattttc acgatatgta aatagtgaaa taggcaaaaa cgccaaaaag
     7681 tagtaagcgc aacatacact aaaccattaa agaatatctc gaccagaatc taacagatat
     7741 acatgttccg ataatgtctg agttaggtga gtattctaaa ttagaaaaca aagagcttag
     7801 aattcccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     7861 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     7921 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     7981 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     8041 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     8101 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     8161 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     8221 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     8281 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     8341 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     8401 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     8461 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     8521 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     8581 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     8641 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     8701 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     8761 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     8821 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     8881 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     8941 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     9001 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     9061 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     9121 ccagtgctgc aatgataccg cgggacccac gctcaccggc tccagattta tcagcaataa
     9181 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     9241 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     9301 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     9361 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     9421 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     9481 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     9541 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     9601 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     9661 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     9721 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     9781 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     9841 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     9901 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     9961 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatc
//