LOCUS       sequence-64801-e        8442 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8442)
  AUTHORS   Finney-Manchester SP, Maheshri N
  TITLE     Harnessing mutagenic homologous recombination for targeted 
            mutagenesis in vivo by TaGTEAM.
  JOURNAL   Nucleic Acids Res. 2013 Mar 7.
  PUBMED    23470991
REFERENCE   2  (bases 1 to 8442)
  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: "Nucleic 
            Acids Res. 2013 Mar 7."
COMMENT     Sequence Label: PRS415-Gal1pr-sctetR-FokI
FEATURES             Location/Qualifiers
     source          1..8442
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(663..1757)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(1770..2174)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     primer_bind     complement(2611..2632)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3006..3447
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    3006..3123
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3418..3441
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             3468..3488
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3489..4103
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="SRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVK
                     NKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRP
                     TEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTD
                     SMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG"
     primer_bind     complement(3559..3578)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5668..5688)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5668..5686)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5707..5723)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5707..5723)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5720..5742)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5731..5747
                     /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(5755..5761,5762..5779,5780..5785))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(5938..5955)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6109..6697)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6189..6208)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6868..7659,7660..7728))
                     /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     7491..7510
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7729..7833)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    7870..8373
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     8415..8433
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatcga ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc
      241 accattatgg gaaatggttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca
      301 ttgagtgttt tttatttgtt gtattttttt ttttttagag aaaatcctcc aatatcaaat
      361 taggaatcgt agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc
      421 ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc
      481 aatttgctta cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt
      541 agattgcgta tatagtttcg tctaccctat gaacatattc cattttgtaa tttcgtgtcg
      601 tttctattat gaatttcatt tataaagttt atgtacaaat atcataaaaa aagagaatct
      661 ttttaagcaa ggattttctt aacttcttcg gcgacagcat caccgacttc ggtggtactg
      721 ttggaaccac ctaaatcacc agttctgata cctgcatcca aaaccttttt aactgcatct
      781 tcaatggcct taccttcttc aggcaagttc aatgacaatt tcaacatcat tgcagcagac
      841 aagatagtgg cgatagggtc aaccttattc tttggcaaat ctggagcaga accgtggcat
      901 ggttcgtaca aaccaaatgc ggtgttcttg tctggcaaag aggccaagga cgcagatggc
      961 aacaaaccca aggaacctgg gataacggag gcttcatcgg agatgatatc accaaacatg
     1021 ttgctggtga ttataatacc atttaggtgg gttgggttct taactaggat catggcggca
     1081 gaatcaatca attgatgttg aaccttcaat gtagggaatt cgttcttgat ggtttcctcc
     1141 acagtttttc tccataatct tgaagaggcc aaaacattag ctttatccaa ggaccaaata
     1201 ggcaatggtg gctcatgttg tagggccatg aaagcggcca ttcttgtgat tctttgcact
     1261 tctggaacgg tgtattgttc actatcccaa gcgacaccat caccatcgtc ttcctttctc
     1321 ttaccaaagt aaatacctcc cactaattct ctgacaacaa cgaagtcagt acctttagca
     1381 aattgtggct tgattggaga taagtctaaa agagagtcgg atgcaaagtt acatggtctt
     1441 aagttggcgt acaattgaag ttctttacgg atttttagta aaccttgttc aggtctaaca
     1501 ctaccggtac cccatttagg accacccaca gcacctaaca aaacggcatc aaccttcttg
     1561 gaggcttcca gcgcctcatc tggaagtggg acacctgtag catcgatagc agcaccacca
     1621 attaaatgat tttcgaaatc gaacttgaca ttggaacgaa catcagaaat agctttaaga
     1681 accttaatgg cttcggctgt gatttcttga ccaacgtggt cacctggcaa aacgacgatc
     1741 ttcttagggg cagacatagg ggcagacatt agaatggtat atccttgaaa tatatatata
     1801 tattgctgaa atgtaaaagg taagaaaagt tagaaagtaa gacgattgct aaccacctat
     1861 tggaaaaaac aataggtcct taaataatat tgtcaacttc aagtattgtg atgcaagcat
     1921 ttagtcatga acgcttctct attctatatg aaaagccggt tccggcctct cacctttcct
     1981 ttttctccca atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca
     2041 aaaaatttcc agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat
     2101 gttgaggaaa aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga
     2161 gtattcccac agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg
     2221 gccaaacaac caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt
     2281 ttgaacacac atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg
     2341 atgtaattgt tgggattcca tttttaataa ggcaataata ttaggtatgt ggatatacta
     2401 gaagttctcc tcgaccgtcg atatgcggtg tgaaataccg cacagatgcg taaggagaaa
     2461 ataccgcatc aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt
     2521 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     2581 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     2641 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     2701 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     2761 cctaaaggga gcccccgatt tagagcttga cggggaaagt cgagcaaaaa ttcttacttt
     2821 ttttttggat ggacgcaaag aagtttaata atcatattac atggcattac caccatatac
     2881 atatccatat ctaatcttac ttatatgttg tggaaatgta aagagcccca ttatcttagc
     2941 ctaaaaaaac cttctctttg gaactttcag taatacgctt aactgctcat tgctatattg
     3001 aagtacggat tagaagccgc cgagcgggcg acagccctcc gacggaagac tctcctccgt
     3061 gcgtcctcgt cttcaccggt cgcgttcctg aaacgcagat gtgcctcgcg ccgcactgct
     3121 ccgaacaata aagattctac aatactagct tttatggtta tgaagaggaa aaattggcag
     3181 taacctggcc ccacaaacct tcaaattaac gaatcaaatt aacaaccata ggatgataat
     3241 gcgattagtt ttttagcctt atttctgggg taattaatca gcgaagcgat gatttttgat
     3301 ctattaacag atatataaat ggaaaagctg cataaccact ttaactaata ctttcaacat
     3361 tttcagtttg tattacttct tattcaaatg tcataaaagt atcaacaaaa aattgttaat
     3421 atacctctat actttaacgt caaggagaaa aaactataaa caaaatgccg aaaaaaaaac
     3481 gcaaagtgtc tagattagat aaaagtaaag tgattaacag cgcattagag ctgcttaatg
     3541 aggtcggaat cgaaggttta acaacccgta aactcgccca gaagcttggt gtagagcagc
     3601 ctacattgta ttggcatgta aaaaataagc gggctttgct cgacgcctta gccattgaga
     3661 tgttagatag gcaccatact cacttttgcc ctttagaagg ggaaagctgg caagattttt
     3721 tacgtaataa cgctaaaagt tttagatgtg ctttactaag tcatcgcgat ggagcaaaag
     3781 tacatttagg tacacggcct acagaaaaac agtatgaaac tctcgaaaat caattagcct
     3841 ttttatgcca acaaggtttt tcactagaga acgcgttata tgcactcagc gctgtggggc
     3901 attttacctt aggttgcgta ttggaagatc aagagcatca agtcgctaaa gaagaaaggg
     3961 aaacacctac tactgatagt atgccgccat tattacgaca agctatcgaa ttatttgatc
     4021 accaaggtgc agagccagcc ttcttattcg gccttgaatt gatcatatgc ggattagaaa
     4081 aacaacttaa atgtgaaagt ggggactcag gaggcggtgg atccggtgga ggcggttctg
     4141 gcggtggagg ctctggaggt ggcggatcgg gcggaggtgg ctctagatct cgtctggaca
     4201 agagcaaagt cataaactct gctctggaat tactcaatga agtcggtatc gaaggcctga
     4261 cgacaaggaa actcgctcaa aagctgggag ttgagcagcc taccctgtac tggcacgtga
     4321 agaacaagcg ggccctgctc gatgccctgg caatcgagat gctggacagg catcataccc
     4381 acttctgccc cctggaaggc gagtcatggc aagactttct gcggaacaac gccaagtcat
     4441 tccgctgtgc tctcctctca catcgcgacg gggctaaagt gcatctcggc acccgcccaa
     4501 cagagaaaca gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag caaggcttct
     4561 ccctggagaa cgcactgtac gctctgtccg ccgtgggcca ctttacactg ggctgcgtat
     4621 tggaggatca ggagcatcaa gtagcaaaag aggaaagaga gacacctacc accgattcta
     4681 tgcccccact tctgagacaa gcaattgagc tgttcgacca tcagggagcc gaacctgcct
     4741 tccttttcgg cctggaacta atcatatgtg gcctggagaa acagctaaag tgcgaaagcg
     4801 gcgacgtcga cggtgctggt ttaattaacc agctggtgaa gagcgagctg gaggagaaga
     4861 agtccgagct gcggcacaag ctgaagtacg tgccccacga gtacatcgag ctgatcgaga
     4921 tcgccaggaa cagcacccag gaccgcatcc tggagatgaa ggtgatggag ttcttcatga
     4981 aggtgtacgg ctacagggga aagcacctgg gcggaagcag aaagcctgac ggcgccatct
     5041 atacagtggg cagccccatc gattacggcg tgatcgtgga cacaaaggcc tacagcggcg
     5101 gctacaatct gcctatcggc caggccgacg agatggagag atacgtggag gagaaccaga
     5161 cccggaataa gcacctcaac cccaacgagt ggtggaaggt gtaccctagc agcgtgaccg
     5221 agttcaagtt cctgttcgtg agcggccact tcaagggcaa ctacaaggcc cagctgacca
     5281 ggctgaacca catcaccaac tgcaatggcg ccgtgctgag cgtggaggag ctgctgatcg
     5341 gcggcgagat gatcaaagcc ggcaccctga cactggagga ggtgcggcgc aagttcaaca
     5401 acggcgagat caacttcaga tcttgataac tcgacgttca tgtgcggccg ctctgctttt
     5461 gtgcgcgtat gtttatgtat gtacctctct ctctatttct atttttaaac caccctctca
     5521 ataaaataaa aataataaag tatttttaag gaaaagacgt gtttaagcac tgactttatc
     5581 tactttttgt acgttttcat tgatataatg tgttttgtct ctcccttttc tacgaaaatt
     5641 tcaaaaattg agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     5701 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5761 aggagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgaggt aactcacatt
     5821 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5881 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5941 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     6001 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     6061 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     6121 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     6181 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     6241 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     6301 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     6361 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     6421 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     6481 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     6541 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6601 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6661 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6721 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6781 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6841 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6901 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6961 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     7021 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     7081 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     7141 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     7201 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     7261 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     7321 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     7381 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     7441 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     7501 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     7561 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7621 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7681 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7741 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7801 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctgg
     7861 gtccttttca tcacgtgcta taaaaataat tataatttaa attttttaat ataaatatat
     7921 aaattaaaaa tagaaagtaa aaaaagaaat taaagaaaaa atagtttttg ttttccgaag
     7981 atgtaaaaga ctctaggggg atcgccaaca aatactacct tttatcttgc tcttcctgct
     8041 ctcaggtatt aatgccgaat tgtttcatct tgtctgtgta gaagaccaca cacgaaaatc
     8101 ctgtgatttt acattttact tatcgttaat cgaatgtata tctatttaat ctgcttttct
     8161 tgtctaataa atatatatgt aaagtacgct ttttgttgaa attttttaaa cctttgttta
     8221 tttttttttc ttcattccgt aactcttcta ccttctttat ttactttcta aaatccaaat
     8281 acaaaacata aaaataaata aacacagagt aaattcccaa attattccat cattaaaaga
     8341 tacgaggcgc gtgtaagtta caggcaagcg atccgtccta agaaaccatt attatcatga
     8401 cattaaccta taaaaatagg cgtatcacga ggccctttcg tc
//
