LOCUS       sequence-44897-e        7498 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 7498)
  AUTHORS   Zhang Y, Zhang F, Li X, Baller JA, Qi Y, Starker CG, Bogdanove AJ, 
            Voytas DF
  TITLE     TALENs enable efficient plant genome engineering.
  JOURNAL   Plant Physiol. 2012 Nov 2.
  PUBMED    23124327
REFERENCE   2  (bases 1 to 7498)
  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: "Plant 
            Physiol. 2012 Nov 2."
COMMENT     Sequence Label: pZHY501
FEATURES             Location/Qualifiers
     source          1..7498
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             16..36
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             37..63
                     /codon_start=1
                     /product="epitope tag from the baculovirus Autographa 
                     californica GP64 envelope fusion protein"
                     /label=AcV5 tag
                     /translation="SWKDASGWS"
     CDS             complement(370..777)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG
                     TRQCIARCMHCLHCGHAATFCNLHKLACVQL"
     primer_bind     627..649
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     641..658
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     642..658
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        668..686
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(734..752)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(773..789)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(773..789)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(786..808)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    797..813
                     /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(821..827,828..845,846..851))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    866..887
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             1073..1660
                     /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      1683..1935
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     promoter        complement(1977..1995)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     terminator      complement(1997..2036)
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     promoter        2331..2735
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             2748..3842
                     /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    4271..4774
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        4816..4920
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5988..6576
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6477..6496
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6730..6747
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      6848..6942
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        6949..6967
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        7044..7442
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
ORIGIN
        1 atggcttcct cccctccaaa gaaaaagaga aaggttagtt ggaaggacgc aagtggttgg
       61 tctagaatgg tggatctacg cacgctcggc tacagtcagc agcagcaaga gaagatcaaa
      121 ccgaaggtgc gttcgacagt ggcgcagcac cacgaggcac tggtgggcca tgggtttaca
      181 cacgcgcaca tcgttgcgct cagccaacac ccggcagcgt tagggaccgt cgctgtcacg
      241 tatcagcaca taatcacggc gttgccagag gcgacacacg aagacatcgt tggcgtcggc
      301 aaacagtggt ccggcgcacg cgccctggag gccttgctca cggatgcggg ggagttgaga
      361 ggtccgccgt tacagttgga cacaggccaa cttgtgaaga ttgcaaaacg tggcggcgtg
      421 accgcaatgg aggcagtgca tgcatcgcgc aatgcactga cgggtgcccc cctggagacg
      481 ggcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg
      541 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
      601 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
      661 gcgcgcgtaa tacgactcac tatagggcga attgggtacc gggccccccc tcgaggtcct
      721 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc
      781 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
      841 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
      901 cactgcccgc tttccaccgg tcgtctccaa cgaccacctc gtcgccttgg cctgcctcgg
      961 cggacgtcct gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag
     1021 aagagtcaat cgccgtattg gcgaacgcac gtcccatcgc gttgccggat cccagctagt
     1081 gaaatctgaa ttggaagaga agaaatctga acttagacat aaattgaaat atgtgccaca
     1141 tgaatatatt gaattgattg aaatcgcaag aaattcaact caggatagaa tccttgaaat
     1201 gaaggtgatg gagttcttta tgaaggttta tggttatcgt ggtaaacatt tgggtggatc
     1261 aaggaaacca gacggagcaa tttatactgt cggatctcct attgattacg gtgtgatcgt
     1321 tgatactaag gcatattcag gaggttataa tcttccaatt ggtcaagcag atgaaatgca
     1381 aagatatgtc gaagagaatc aaacaagaaa caagcatatc aaccctaatg aatggtggaa
     1441 agtctatcca tcttcagtaa cagaatttaa gttcttgttt gtgagtggtc atttcaaagg
     1501 aaactacaaa gctcagctta caagattgaa tcatatcact aattgtaatg gagctgttct
     1561 tagtgtagaa gagcttttga ttggtggaga aatgattaaa gctggtacat tgacacttga
     1621 ggaagtgaga aggaaattta ataacggtga gataaacttt taataggagc tcgaatttcc
     1681 ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg
     1741 cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat
     1801 gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat
     1861 acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat
     1921 ctatgttact agatcgtggg cccacattcc tgtagaactc ggtgagactt ttgttccctt
     1981 tagtgagggt taattgtcca aaaaaaaagg ctccaaaagg agcctttaat tgtttcggtt
     2041 tatgcttctc cttacgcatc tgtgcggtat ttcacaccgc atatcgacgg tcgaggagaa
     2101 cttctagtat atccacatac ctaatattat tgccttatta aaaatggaat cccaacaatt
     2161 acatcaaaat ccacattctc ttcaaaatca attgtcctgt acttccttgt tcatgtgtgt
     2221 tcaaaaacgt tatatttata ggataattat actctatttc tcaacaagta attggttgtt
     2281 tggccgagcg gtctaaggcg cctgattcaa gaaatatctt gaccgcagtt aactgtggga
     2341 atactcaggt atcgtaagat gcaagagttc gaatctctta gcaaccatta tttttttcct
     2401 caacataacg agaacacaca ggggcgctat cgcacagaat caaattcgat gactggaaat
     2461 tttttgttaa tttcagaggt cgcctgacgc atataccttt ttcaactgaa aaattgggag
     2521 aaaaaggaaa ggtgagaggc cggaaccggc ttttcatata gaatagagaa gcgttcatga
     2581 ctaaatgctt gcatcacaat acttgaagtt gacaatatta tttaaggacc tattgttttt
     2641 tccaataggt ggttagcaat cgtcttactt tctaactttt cttacctttt acatttcagc
     2701 aatatatata tatatttcaa ggatatacca ttctaatgtc tgcccctatg tctgccccta
     2761 agaagatcgt cgttttgcca ggtgaccacg ttggtcaaga aatcacagcc gaagccatta
     2821 aggttcttaa agctatttct gatgttcgtt ccaatgtcaa gttcgatttc gaaaatcatt
     2881 taattggtgg tgctgctatc gatgctacag gtgtcccact tccagatgag gcgctggaag
     2941 cctccaagaa ggttgatgcc gttttgttag gtgctgtggg tggtcctaaa tggggtaccg
     3001 gtagtgttag acctgaacaa ggtttactaa aaatccgtaa agaacttcaa ttgtacgcca
     3061 acttaagacc atgtaacttt gcatccgact ctcttttaga cttatctcca atcaagccac
     3121 aatttgctaa aggtactgac ttcgttgttg tcagagaatt agtgggaggt atttactttg
     3181 gtaagagaaa ggaagacgat ggtgatggtg tcgcttggga tagtgaacaa tacaccgttc
     3241 cagaagtgca aagaatcaca agaatggccg ctttcatggc cctacaacat gagccaccat
     3301 tgcctatttg gtccttggat aaagctaatg ttttggcctc ttcaagatta tggagaaaaa
     3361 ctgtggagga aaccatcaag aacgaattcc ctacattgaa ggttcaacat caattgattg
     3421 attctgccgc catgatccta gttaagaacc caacccacct aaatggtatt ataatcacca
     3481 gcaacatgtt tggtgatatc atctccgatg aagcctccgt tatcccaggt tccttgggtt
     3541 tgttgccatc tgcgtccttg gcctctttgc cagacaagaa caccgcattt ggtttgtacg
     3601 aaccatgcca cggttctgct ccagatttgc caaagaataa ggttgaccct atcgccacta
     3661 tcttgtctgc tgcaatgatg ttgaaattgt cattgaactt gcctgaagaa ggtaaggcca
     3721 ttgaagatgc agttaaaaag gttttggatg caggtatcag aactggtgat ttaggtggtt
     3781 ccaacagtac caccgaagtc ggtgatgctg tcgccgaaga agttaagaaa atccttgctt
     3841 aaaaagattc tcttttttta tgatatttgt acataaactt tataaatgaa attcataata
     3901 gaaacgacac gaaattacaa aatggaatat gttcataggg tagacgaaac tatatacgca
     3961 atctacatac atttatcaag aaggagaaaa aggaggatag taaaggaata caggtaagca
     4021 aattgatact aatggctcaa cgtgataagg aaaaagaatt gcactttaac attaatattg
     4081 acaaggagga gggcaccaca caaaaagtta ggtgtaacag aaaatcatga aactacgatt
     4141 cctaatttga tattggagga ttttctctaa aaaaaaaaaa atacaacaaa taaaaaacac
     4201 tcaatgacct gaccatttga tgggctcacc aagctcttaa aacgggaatt tatggatctt
     4261 aattaagacg gatcgcttgc ctgtaactta cacgcgcctc gtatctttta atgatggaat
     4321 aatttgggaa tttactctgt gtttatttat ttttatgttt tgtatttgga ttttagaaag
     4381 taaataaaga aggtagaaga gttacggaat gaagaaaaaa aaataaacaa aggtttaaaa
     4441 aatttcaaca aaaagcgtac tttacatata tatttattag acaagaaaag cagattaaat
     4501 agatatacat tcgattaacg ataagtaaaa tgtaaaatca caggattttc gtgtgtggtc
     4561 ttctacacag acaagatgaa acaattcggc attaatacct gagagcagga agagcaagat
     4621 aaaaggtagt atttgttggc gatcccccta gagtctttta catcttcgga aaacaaaaac
     4681 tattttttct ttaatttctt tttttacttt ctatttttaa tttatatatt tatattaaaa
     4741 aatttaaatt ataattattt ttatagcacg tgatgaaaag ccttagggtc aggtggcact
     4801 tttcggggaa atgtgcgatg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4861 tatccgctaa tgagacaata accctgataa atgcttcaat tatattgaaa aaggaagagt
     4921 atgagtatcc aacatttccg tgtcgccctt attccctttt ttgcggcctt ctgccttcct
     4981 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgcc
     5041 cgagtgggtt acattgaact ggatctcaac agcggcaaaa tcttagagag ttttcgcccc
     5101 gaagaacgct tcccaatgat gagcactttc aaagttctgc tatgtggcgc ggtgttatcc
     5161 cgtattgatg ctgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5221 gttgagtatt caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5281 tgtagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatt
     5341 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5401 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5461 cctgtagcaa tggcaacaac tctccgcaaa ctattaactg gcgaactact tactcttgct
     5521 tcacgccagc aactcattga ctggatggag gcggacaaag ttgcaggacc acttctgcgc
     5581 tcggcacttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     5641 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     5701 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     5761 tcactgatta agcattggta actgtcagac gaagggaaat aaatagtagc ccgcctgatg
     5821 tgcgggcttt tttttggtgg tggttaccag tgtcgcttag ctaccgtagg tcctcttagg
     5881 cggtccgatt agcgacatag tcatcaatgc cttgacggca aaatccctta acgtgagttt
     5941 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6001 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6061 ttgccggatc aagagctact aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6121 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6181 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6241 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6301 ggctgaacgg ggggttcgtg ctcacagccc agcttggagc gaacgaccta caccgaactg
     6361 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6421 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6481 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6541 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6601 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     6661 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     6721 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     6781 ctccccgcgc gttggccgat tcattgatgc aggtcaacga catcacctgg ttgcgagcct
     6841 tctgaaggac tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc
     6901 agaacgctcg gttgccgccg ggcgtttttt attggtgaga atcaggcgta atacgactca
     6961 ctatagggcg aatggaactg gacgctcgcc tgtggtcgca gggtggctcg tacgaactta
     7021 ccgcggagac atatcgataa tcacatagct tcaaaatgtt tctactcctt ttttactctt
     7081 ccagattttc tcggactccg cgcatcgccg taccacttca aaacacccaa gcacagcata
     7141 ctaaatttcc cctctttctt cctctagggt gtcgttaatt acccgtacta aaggtttgga
     7201 aaagaaaaaa gagaccgcct cgtttctttt tcttcgtcga aaaaggcaat aaaaattttt
     7261 atcacgtttc tttttcttga aaattttttt tttgattttt ttctctttcg atgacctccc
     7321 attgatattt aagttaataa acggtcttca atttctcaag tttcagtttc atttttcttg
     7381 ttctattaca acttttttta cttcttgctc attagaaaga aagcatagca atctaatcta
     7441 agtttagatc taactcgaga aagatattgt atatatcgta acaataggag gttcaaca
//
