LOCUS       sequence-64793-e        8698 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 8698)
  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 8698)
  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-Mag1-sctetR
FEATURES             Location/Qualifiers
     source          1..8698
                     /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             4385..4999
                     /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(4455..4474)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5924..5944)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5924..5942)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5963..5979)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5963..5979)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5976..5998)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5987..6003
                     /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(6011..6017,6018..6035,6036..6041))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(6194..6211)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6365..6953)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6445..6464)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7124..7915,7916..7984))
                     /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     7747..7766
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7985..8089)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    8126..8629
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     8671..8689
                     /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 aaacggatcc actagtaaca aaatgaaact
     3481 aaaaagggag tatgatgagt taataaaagc agacgctgtt aaggaaatag caaaagaatt
     3541 agggtctcga cctctagagg ttgctcttcc tgagaaatat attgctagac atgaagaaaa
     3601 gttcaatatg gcttgcgaac acattttaga gaaagatcca tcactttttc ccatacttaa
     3661 gaataatgaa tttacgttgt acttgaagga gactcaagtc cctaatacac tcgaagatta
     3721 ttttattagg cttgcaagca caattttgtc tcaacagatc agtggccaag cagctgaaag
     3781 catcaaggca agggttgtca gtctttatgg cggtgcattt cctgattaca aaatcctttt
     3841 cgaagacttc aaagacccag caaaatgtgc agaaatcgca aaatgtggat tgagtaaaag
     3901 gaaaatgata tatctagagt ctcttgctgt ctattttact gaaaaatata aggatatcga
     3961 aaagctcttc ggtcaaaaag ataatgatga ggaagtgatt gaaagtttag ttacgaatgt
     4021 aaaaggtata ggcccatgga gtgccaaaat gttcttgatc tccggattga aaagaatgga
     4081 tgtatttgct cctgaagatc taggtattgc taggggtttt tcaaaatacc tttcagataa
     4141 gccagaattg gaaaaagaat taatgcgtga aagaaaagta gttaaaaaga gtaagattaa
     4201 gcataagaaa tacaactgga aaatatatga cgacgacata atggaaaaat gctctgaaac
     4261 attttctccg tataggtctg tgtttatgtt catactttgg aggctcgcga gcacaaatac
     4321 agatgccatg atgaaggcag aagaaaattt cgtgaaatcc gtcgacggtg ctggtttaat
     4381 taactctaga ttagataaaa gtaaagtgat taacagcgca ttagagctgc ttaatgaggt
     4441 cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag cttggtgtag agcagcctac
     4501 attgtattgg catgtaaaaa ataagcgggc tttgctcgac gccttagcca ttgagatgtt
     4561 agataggcac catactcact tttgcccttt agaaggggaa agctggcaag attttttacg
     4621 taataacgct aaaagtttta gatgtgcttt actaagtcat cgcgatggag caaaagtaca
     4681 tttaggtaca cggcctacag aaaaacagta tgaaactctc gaaaatcaat tagccttttt
     4741 atgccaacaa ggtttttcac tagagaacgc gttatatgca ctcagcgctg tggggcattt
     4801 taccttaggt tgcgtattgg aagatcaaga gcatcaagtc gctaaagaag aaagggaaac
     4861 acctactact gatagtatgc cgccattatt acgacaagct atcgaattat ttgatcacca
     4921 aggtgcagag ccagccttct tattcggcct tgaattgatc atatgcggat tagaaaaaca
     4981 acttaaatgt gaaagtgggg actcaggagg cggtggatcc ggtggaggcg gttctggcgg
     5041 tggaggctct ggaggtggcg gatcgggcgg aggtggctct agatctcgtc tggacaagag
     5101 caaagtcata aactctgctc tggaattact caatgaagtc ggtatcgaag gcctgacgac
     5161 aaggaaactc gctcaaaagc tgggagttga gcagcctacc ctgtactggc acgtgaagaa
     5221 caagcgggcc ctgctcgatg ccctggcaat cgagatgctg gacaggcatc atacccactt
     5281 ctgccccctg gaaggcgagt catggcaaga ctttctgcgg aacaacgcca agtcattccg
     5341 ctgtgctctc ctctcacatc gcgacggggc taaagtgcat ctcggcaccc gcccaacaga
     5401 gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg tgtcagcaag gcttctccct
     5461 ggagaacgca ctgtacgctc tgtccgccgt gggccacttt acactgggct gcgtattgga
     5521 ggatcaggag catcaagtag caaaagagga aagagagaca cctaccaccg attctatgcc
     5581 cccacttctg agacaagcaa ttgagctgtt cgaccatcag ggagccgaac ctgccttcct
     5641 tttcggcctg gaactaatca tatgtggcct ggagaaacag ctaaagtgcg aaagcggcga
     5701 ctaataatct gcttttgtgc gcgtatgttt atgtatgtac ctctctctct atttctattt
     5761 ttaaaccacc ctctcaataa aataaaaata ataaagtatt tttaaggaaa agacgtgttt
     5821 aagcactgac tttatctact ttttgtacgt tttcattgat ataatgtgtt ttgtctctcc
     5881 cttttctacg aaaatttcaa aaattgagct ccagcttttg ttccctttag tgagggttaa
     5941 ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     6001 caattccaca caacatagga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     6061 tgaggtaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     6121 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     6181 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     6241 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     6301 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     6361 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     6421 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     6481 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     6541 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     6601 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     6661 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     6721 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     6781 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     6841 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     6901 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     6961 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     7021 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     7081 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     7141 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     7201 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     7261 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     7321 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     7381 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     7441 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     7501 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     7561 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     7621 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     7681 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     7741 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     7801 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     7861 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     7921 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     7981 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     8041 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     8101 gaaaagtgcc acctgggtcc ttttcatcac gtgctataaa aataattata atttaaattt
     8161 tttaatataa atatataaat taaaaataga aagtaaaaaa agaaattaaa gaaaaaatag
     8221 tttttgtttt ccgaagatgt aaaagactct agggggatcg ccaacaaata ctacctttta
     8281 tcttgctctt cctgctctca ggtattaatg ccgaattgtt tcatcttgtc tgtgtagaag
     8341 accacacacg aaaatcctgt gattttacat tttacttatc gttaatcgaa tgtatatcta
     8401 tttaatctgc ttttcttgtc taataaatat atatgtaaag tacgcttttt gttgaaattt
     8461 tttaaacctt tgtttatttt tttttcttca ttccgtaact cttctacctt ctttatttac
     8521 tttctaaaat ccaaatacaa aacataaaaa taaataaaca cagagtaaat tcccaaatta
     8581 ttccatcatt aaaagatacg aggcgcgtgt aagttacagg caagcgatcc gtcctaagaa
     8641 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
