LOCUS       sequence-14200-e        8467 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 8467)
  AUTHORS   Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller 
            JA, Somia NV, Bogdanove AJ, Voytas DF
  TITLE     Efficient design and assembly of custom TALEN and other TAL 
            effector-based constructs for DNA targeting.
  JOURNAL   Nucleic Acids Res. 2011 Jul;39(12):e82. doi: 10.1093/nar/gkr218
  PUBMED    21493687
REFERENCE   2  (bases 1 to 8467)
  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."; date: "2011-07"; volume: "39(12):e82. doi"; pages: " 
            10.1093/nar/gkr21"
COMMENT     Sequence Label: pTAL4
FEATURES             Location/Qualifiers
     source          1..8467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      35..129
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        136..154
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        231..629
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             701..721
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             722..748
                     /codon_start=1
                     /product="epitope tag from the baculovirus Autographa 
                     californica GP64 envelope fusion protein"
                     /label=AcV5 tag
                     /translation="SWKDASGWS"
     CDS             complement(1514..1921)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG
                     TRQCIARCMHCLHCGHAATFCNLHKLACVQL"
     primer_bind     1771..1793
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1785..1802
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1786..1802
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1812..1830
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1878..1896)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1917..1933)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1917..1933)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1930..1952)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1941..1957
                     /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(1965..1971,1972..1989,1990..1995))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2010..2031
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             2727..3314
                     /codon_start=1
                     /product="nonspecific DNA cleavage domain of the FokI 
                     endonuclease (Li et al., 1992)"
                     /label=FokI cleavage domain
                     /note="must dimerize to be catalytically active"
                     /translation="QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKV
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     terminator      3337..3589
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     promoter        complement(3631..3649)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     terminator      complement(3651..3690)
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     promoter        3985..4389
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             4402..5496
                     /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"
     misc_feature    5925..6428
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        6470..6574
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7642..8230
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8131..8150
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8384..8401
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 tcaacgacat cacctggttg cgagccttct gaaggactcc tgttgataga tccagtaatg
       61 acctcagaac tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatt
      121 ggtgagaatc aggcgtaata cgactcacta tagggcgaat ggaactggac gctcgcctgt
      181 ggtcgcaggg tggctcgtac gaacttaccg cggagacata tcgataatca catagcttca
      241 aaatgtttct actccttttt tactcttcca gattttctcg gactccgcgc atcgccgtac
      301 cacttcaaaa cacccaagca cagcatacta aatttcccct ctttcttcct ctagggtgtc
      361 gttaattacc cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt ttctttttct
      421 tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt ttcttgaaaa tttttttttt
      481 gatttttttc tctttcgatg acctcccatt gatatttaag ttaataaacg gtcttcaatt
      541 tctcaagttt cagtttcatt tttcttgttc tattacaact ttttttactt cttgctcatt
      601 agaaagaaag catagcaatc taatctaagt ttagatctaa ctcgagaaag atattgtata
      661 tatcgtaaca ataggaggtt caacaatggc ttcctcccct ccaaagaaaa agagaaaggt
      721 tagttggaag gacgcaagtg gttggtctag aatgcatgcg gatcccattc gtccgcgcag
      781 gccaagtcct gcccgcgagc ttctgcccgg accccaaccg gatagggttc agccgactgc
      841 agatcgtggg gtgtctgcgc ctgctggcag ccctctggat ggcttgcccg ctcggcggac
      901 ggtgtcccgg acccggctgc catctccccc tgcgccctca cctgcgttct cggcgggcag
      961 cttcagcgat ctgctccgtc cgttcgatcc gtcgcttctt gatacatcgc ttcttgattc
     1021 gatgcctgcc gtcggcacgc cgcatacagc ggctgcccca gcagagtggg atgaggcgca
     1081 atcggctctg cgtgcagccg atgacccgcc acccaccgtg cgtgtcgctg tcactgccgc
     1141 gcggccgccg cgcgccaagc cggccccgcg acggcgtgct gcgcaaccct ccgacgcttc
     1201 gccggccgcg caggtggatc tacgcacgct cggctacagt cagcagcagc aagagaagat
     1261 caaaccgaag gtgcgttcga cagtggcgca gcaccacgag gcactggtgg gccatgggtt
     1321 tacacacgcg cacatcgttg cgctcagcca acacccggca gcgttaggga ccgtcgctgt
     1381 cacgtatcag cacataatca cggcgttgcc agaggcgaca cacgaagaca tcgttggcgt
     1441 cggcaaacag tggtccggcg cacgcgccct ggaggccttg ctcacggatg cgggggagtt
     1501 gagaggtccg ccgttacagt tggacacagg ccaacttgtg aagattgcaa aacgtggcgg
     1561 cgtgaccgca atggaggcag tgcatgcatc gcgcaatgca ctgacgggtg cccccctgga
     1621 gacgggcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag
     1681 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     1741 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     1801 gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccgggccc cccctcgagg
     1861 tcctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
     1921 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     1981 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     2041 cgctcactgc ccgctttcca ccggtcgtct ccaacgacca cctcgtcgcc ttggcctgcc
     2101 tcggcggacg tcctgccatg gatgcagtga aaaagggatt gccgcacgcg ccggaattga
     2161 tcagaagagt caatcgccgt attggcgaac gcacgtccca tcgcgttgcc gactacgcgc
     2221 aagtggttcg cgtgctggag tttttccagt gccactccca cccagcgtac gcatttgatg
     2281 aggccatgac gcagttcggg atgagcagga acgggttggt acagctcttt cgcagagtgg
     2341 gcgtcaccga actcgaagcc cgcggtggaa cgctcccccc agcctcgcag cgttgggacc
     2401 gtatcctcca ggcatcaggg atgaaaaggg ccaaaccgtc ccctacttca gctcaaacac
     2461 cggatcaggc gtctttgcat gcattcgccg attcgctgga gcgtgacctt gatgcgccca
     2521 gcccaatgca cgagggagat cagacgcggg caagcagccg taaacggtcc cgatcggatc
     2581 gtgctgtcac cggcccctcc gcacagcagg ctgtcgaggt gcgcgttccc gaacagcgcg
     2641 atgcgctgca tttgcccctc agctggaggg taaaacgccc gcgtaccagg atctggggcg
     2701 gcctcccgga tccgatatct agatcccagc tagtgaaatc tgaattggaa gagaagaaat
     2761 ctgaacttag acataaattg aaatatgtgc cacatgaata tattgaattg attgaaatcg
     2821 caagaaattc aactcaggat agaatccttg aaatgaaggt gatggagttc tttatgaagg
     2881 tttatggtta tcgtggtaaa catttgggtg gatcaaggaa accagacgga gcaatttata
     2941 ctgtcggatc tcctattgat tacggtgtga tcgttgatac taaggcatat tcaggaggtt
     3001 ataatcttcc aattggtcaa gcagatgaaa tgcaaagata tgtcgaagag aatcaaacaa
     3061 gaaacaagca tatcaaccct aatgaatggt ggaaagtcta tccatcttca gtaacagaat
     3121 ttaagttctt gtttgtgagt ggtcatttca aaggaaacta caaagctcag cttacaagat
     3181 tgaatcatat cactaattgt aatggagctg ttcttagtgt agaagagctt ttgattggtg
     3241 gagaaatgat taaagctggt acattgacac ttgaggaagt gagaaggaaa tttaataacg
     3301 gtgagataaa cttttaatag gagctcgaat ttccccgatc gttcaaacat ttggcaataa
     3361 agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg
     3421 aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt
     3481 tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc
     3541 gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg tgggcccaca
     3601 ttcctgtaga actcggtgag acttttgttc cctttagtga gggttaattg tccaaaaaaa
     3661 aaggctccaa aaggagcctt taattgtttc ggtttatgct tctccttacg catctgtgcg
     3721 gtatttcaca ccgcatatcg acggtcgagg agaacttcta gtatatccac atacctaata
     3781 ttattgcctt attaaaaatg gaatcccaac aattacatca aaatccacat tctcttcaaa
     3841 atcaattgtc ctgtacttcc ttgttcatgt gtgttcaaaa acgttatatt tataggataa
     3901 ttatactcta tttctcaaca agtaattggt tgtttggccg agcggtctaa ggcgcctgat
     3961 tcaagaaata tcttgaccgc agttaactgt gggaatactc aggtatcgta agatgcaaga
     4021 gttcgaatct cttagcaacc attatttttt tcctcaacat aacgagaaca cacaggggcg
     4081 ctatcgcaca gaatcaaatt cgatgactgg aaattttttg ttaatttcag aggtcgcctg
     4141 acgcatatac ctttttcaac tgaaaaattg ggagaaaaag gaaaggtgag aggccggaac
     4201 cggcttttca tatagaatag agaagcgttc atgactaaat gcttgcatca caatacttga
     4261 agttgacaat attatttaag gacctattgt tttttccaat aggtggttag caatcgtctt
     4321 actttctaac ttttcttacc ttttacattt cagcaatata tatatatatt tcaaggatat
     4381 accattctaa tgtctgcccc tatgtctgcc cctaagaaga tcgtcgtttt gccaggtgac
     4441 cacgttggtc aagaaatcac agccgaagcc attaaggttc ttaaagctat ttctgatgtt
     4501 cgttccaatg tcaagttcga tttcgaaaat catttaattg gtggtgctgc tatcgatgct
     4561 acaggtgtcc cacttccaga tgaggcgctg gaagcctcca agaaggttga tgccgttttg
     4621 ttaggtgctg tgggtggtcc taaatggggt accggtagtg ttagacctga acaaggttta
     4681 ctaaaaatcc gtaaagaact tcaattgtac gccaacttaa gaccatgtaa ctttgcatcc
     4741 gactctcttt tagacttatc tccaatcaag ccacaatttg ctaaaggtac tgacttcgtt
     4801 gttgtcagag aattagtggg aggtatttac tttggtaaga gaaaggaaga cgatggtgat
     4861 ggtgtcgctt gggatagtga acaatacacc gttccagaag tgcaaagaat cacaagaatg
     4921 gccgctttca tggccctaca acatgagcca ccattgccta tttggtcctt ggataaagct
     4981 aatgttttgg cctcttcaag attatggaga aaaactgtgg aggaaaccat caagaacgaa
     5041 ttccctacat tgaaggttca acatcaattg attgattctg ccgccatgat cctagttaag
     5101 aacccaaccc acctaaatgg tattataatc accagcaaca tgtttggtga tatcatctcc
     5161 gatgaagcct ccgttatccc aggttccttg ggtttgttgc catctgcgtc cttggcctct
     5221 ttgccagaca agaacaccgc atttggtttg tacgaaccat gccacggttc tgctccagat
     5281 ttgccaaaga ataaggttga ccctatcgcc actatcttgt ctgctgcaat gatgttgaaa
     5341 ttgtcattga acttgcctga agaaggtaag gccattgaag atgcagttaa aaaggttttg
     5401 gatgcaggta tcagaactgg tgatttaggt ggttccaaca gtaccaccga agtcggtgat
     5461 gctgtcgccg aagaagttaa gaaaatcctt gcttaaaaag attctctttt tttatgatat
     5521 ttgtacataa actttataaa tgaaattcat aatagaaacg acacgaaatt acaaaatgga
     5581 atatgttcat agggtagacg aaactatata cgcaatctac atacatttat caagaaggag
     5641 aaaaaggagg atagtaaagg aatacaggta agcaaattga tactaatggc tcaacgtgat
     5701 aaggaaaaag aattgcactt taacattaat attgacaagg aggagggcac cacacaaaaa
     5761 gttaggtgta acagaaaatc atgaaactac gattcctaat ttgatattgg aggattttct
     5821 ctaaaaaaaa aaaaatacaa caaataaaaa acactcaatg acctgaccat ttgatgggct
     5881 caccaagctc ttaaaacggg aatttatgga tcttaattaa gacggatcgc ttgcctgtaa
     5941 cttacacgcg cctcgtatct tttaatgatg gaataatttg ggaatttact ctgtgtttat
     6001 ttatttttat gttttgtatt tggattttag aaagtaaata aagaaggtag aagagttacg
     6061 gaatgaagaa aaaaaaataa acaaaggttt aaaaaatttc aacaaaaagc gtactttaca
     6121 tatatattta ttagacaaga aaagcagatt aaatagatat acattcgatt aacgataagt
     6181 aaaatgtaaa atcacaggat tttcgtgtgt ggtcttctac acagacaaga tgaaacaatt
     6241 cggcattaat acctgagagc aggaagagca agataaaagg tagtatttgt tggcgatccc
     6301 cctagagtct tttacatctt cggaaaacaa aaactatttt ttctttaatt tcttttttta
     6361 ctttctattt ttaatttata tatttatatt aaaaaattta aattataatt atttttatag
     6421 cacgtgatga aaagccttag ggtcaggtgg cacttttcgg ggaaatgtgc gatgaacccc
     6481 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctaatgagac aataaccctg
     6541 ataaatgctt caattatatt gaaaaaggaa gagtatgagt atccaacatt tccgtgtcgc
     6601 ccttattccc ttttttgcgg ccttctgcct tcctgttttt gctcacccag aaacgctggt
     6661 gaaagtaaaa gatgctgaag atcagttggg tgcccgagtg ggttacattg aactggatct
     6721 caacagcggc aaaatcttag agagttttcg ccccgaagaa cgcttcccaa tgatgagcac
     6781 tttcaaagtt ctgctatgtg gcgcggtgtt atcccgtatt gatgctgggc aagagcaact
     6841 cggtcgccgc atacactatt ctcagaatga cttggttgag tattcaccag tcacagaaaa
     6901 gcatcttacg gatggcatga cagtaagaga attatgtagt gctgccataa ccatgagtga
     6961 taacactgcg gccaacttac ttctgacaac gattggagga ccgaaggagc taaccgcttt
     7021 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     7081 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caactctccg
     7141 caaactatta actggcgaac tacttactct tgcttcacgc cagcaactca ttgactggat
     7201 ggaggcggac aaagttgcag gaccacttct gcgctcggca cttccggctg gctggtttat
     7261 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     7321 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     7381 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     7441 agacgaaggg aaataaatag tagcccgcct gatgtgcggg cttttttttg gtggtggtta
     7501 ccagtgtcgc ttagctaccg taggtcctct taggcggtcc gattagcgac atagtcatca
     7561 atgccttgac ggcaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     7621 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     7681 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc tactaactct
     7741 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     7801 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     7861 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     7921 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgctcaca
     7981 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     8041 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     8101 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     8161 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     8221 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     8281 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     8341 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     8401 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcattg
     8461 atgcagg
//
