LOCUS       sequence-44896-e        6561 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 6561)
  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 6561)
  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: pZHY500
FEATURES             Location/Qualifiers
     source          1..6561
                     /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)"
     CDS             complement(2336..2995)
                     /codon_start=1
                     /gene="S. cerevisiae HIS3"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=HIS3
                     /note="yeast auxotrophic marker"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL
                     ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     promoter        complement(2996..3183)
                     /gene="S. cerevisiae HIS3"
                     /label=HIS3 promoter
     misc_feature    3334..3837
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        3879..3983
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5051..5639
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5540..5559
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5793..5810
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      5911..6005
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        6012..6030
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        6107..6505
                     /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 tatgcttagg acaccattga ttaacgctta caatttcctg atgcggtatt ttctccttac
     2101 gcatctgtgc ggtatttcac accgcataga tccgtcgagt tcaagagaaa aaaaaagaaa
     2161 aagcaaaaag aaaaaaggaa agcgcgcctc gttcagaatg acacgtatag aatgatgcat
     2221 taccttgtca tcttcagtat catactgttc gtataaatac ttactgacat tcatagctat
     2281 acatatatac acatgtatat atatcgtatg ctggagcttt aaataatcgg tgtcactaca
     2341 taagaacacc tttggtggag ggaacatcgt tggttccatt gggcgaggtg gcttctctta
     2401 tggcaaccgc aagagccttg aacgcactct cactacggtg atggtcattc ttgcctcgca
     2461 gacaatcaac gtggagggta attctgcttg cctctgcaaa actttcaaga aaatgcggga
     2521 tcatctcgca agagaggtct cctactttct ccctttgcaa accaagttcg acaactgcgt
     2581 atggcctgtt agaaaggtct accaccgctc tggaaagtgc ctcatccaaa ggcgcaaatc
     2641 ctgatccaaa cctttttact ccacgcgcca gtagagcctc tttaaaagcc tgaccgagag
     2701 caatcccgca gtcttcagtg gtgtgatggt cgtctatgtg taagtcacca atgcactcaa
     2761 cgattagcga ccagccggaa tgcttggcca gagcatgtat catgtggtcc agaaacccta
     2821 tacctgtgtg gacgttaatc acttgcgatt gtgtggcctg ttctgctact gcttctgcct
     2881 ctttttctgg gaagatcgag tgctctatcg ctaggggacc accctttaaa gagatagcaa
     2941 tctgaatctt ggtttcattt gtaatacgct ttactagggc tttctgctct gtcatctttg
     3001 ccttcgttta tcttgcctgc tcatttttta gtatattctt cgaagaaatc acattacttt
     3061 atataatgta taattcatta tgtgataatg ccaatcgcta agaaaaaaaa agagtcatcc
     3121 gctaggtgga aaaaaaaaaa tgaaaatcat taccgaggca taaaaaaata tagagtgtac
     3181 tagaggaggc caagagtaat agaaaaagaa aattgcggga aaggactgtg ttatgacttc
     3241 cctgactaat gccgtgttca aacgatacct ggcagtgact cctagcgctc accaagctct
     3301 taaaacggga atttatggat cttaattaag acggatcgct tgcctgtaac ttacacgcgc
     3361 ctcgtatctt ttaatgatgg aataatttgg gaatttactc tgtgtttatt tatttttatg
     3421 ttttgtattt ggattttaga aagtaaataa agaaggtaga agagttacgg aatgaagaaa
     3481 aaaaaataaa caaaggttta aaaaatttca acaaaaagcg tactttacat atatatttat
     3541 tagacaagaa aagcagatta aatagatata cattcgatta acgataagta aaatgtaaaa
     3601 tcacaggatt ttcgtgtgtg gtcttctaca cagacaagat gaaacaattc ggcattaata
     3661 cctgagagca ggaagagcaa gataaaaggt agtatttgtt ggcgatcccc ctagagtctt
     3721 ttacatcttc ggaaaacaaa aactattttt tctttaattt ctttttttac tttctatttt
     3781 taatttatat atttatatta aaaaatttaa attataatta tttttatagc acgtgatgaa
     3841 aagccttagg gtcaggtggc acttttcggg gaaatgtgcg atgaacccct atttgtttat
     3901 ttttctaaat acattcaaat atgtatccgc taatgagaca ataaccctga taaatgcttc
     3961 aattatattg aaaaaggaag agtatgagta tccaacattt ccgtgtcgcc cttattccct
     4021 tttttgcggc cttctgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     4081 atgctgaaga tcagttgggt gcccgagtgg gttacattga actggatctc aacagcggca
     4141 aaatcttaga gagttttcgc cccgaagaac gcttcccaat gatgagcact ttcaaagttc
     4201 tgctatgtgg cgcggtgtta tcccgtattg atgctgggca agagcaactc ggtcgccgca
     4261 tacactattc tcagaatgac ttggttgagt attcaccagt cacagaaaag catcttacgg
     4321 atggcatgac agtaagagaa ttatgtagtg ctgccataac catgagtgat aacactgcgg
     4381 ccaacttact tctgacaacg attggaggac cgaaggagct aaccgctttt ttgcacaaca
     4441 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4501 acgacgagcg tgacaccacg atgcctgtag caatggcaac aactctccgc aaactattaa
     4561 ctggcgaact acttactctt gcttcacgcc agcaactcat tgactggatg gaggcggaca
     4621 aagttgcagg accacttctg cgctcggcac ttccggctgg ctggtttatt gctgataaat
     4681 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4741 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4801 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gacgaaggga
     4861 aataaatagt agcccgcctg atgtgcgggc ttttttttgg tggtggttac cagtgtcgct
     4921 tagctaccgt aggtcctctt aggcggtccg attagcgaca tagtcatcaa tgccttgacg
     4981 gcaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     5041 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     5101 caccgctacc agcggtggtt tgtttgccgg atcaagagct actaactctt tttccgaagg
     5161 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     5221 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     5281 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     5341 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgctcacag cccagcttgg
     5401 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     5461 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5521 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5581 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5641 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     5701 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     5761 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     5821 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattga tgcaggtcaa
     5881 cgacatcacc tggttgcgag ccttctgaag gactcctgtt gatagatcca gtaatgacct
     5941 cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtg
     6001 agaatcaggc gtaatacgac tcactatagg gcgaatggaa ctggacgctc gcctgtggtc
     6061 gcagggtggc tcgtacgaac ttaccgcgga gacatatcga taatcacata gcttcaaaat
     6121 gtttctactc cttttttact cttccagatt ttctcggact ccgcgcatcg ccgtaccact
     6181 tcaaaacacc caagcacagc atactaaatt tcccctcttt cttcctctag ggtgtcgtta
     6241 attacccgta ctaaaggttt ggaaaagaaa aaagagaccg cctcgtttct ttttcttcgt
     6301 cgaaaaaggc aataaaaatt tttatcacgt ttctttttct tgaaaatttt ttttttgatt
     6361 tttttctctt tcgatgacct cccattgata tttaagttaa taaacggtct tcaatttctc
     6421 aagtttcagt ttcatttttc ttgttctatt acaacttttt ttacttcttg ctcattagaa
     6481 agaaagcata gcaatctaat ctaagtttag atctaactcg agaaagatat tgtatatatc
     6541 gtaacaatag gaggttcaac a
//
