LOCUS       sequence-70389-e        5798 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of TALEN proteins with FokI-RRR domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5798)
  AUTHORS   Hu R, Wallace J, Dahlem TJ, Grunwald DJ, O'Connell RM
  TITLE     Targeting human microRNA genes using engineered Tal-effector 
            nucleases (TALENs).
  JOURNAL   PLoS One. 2013 May 7;8(5):e63074. doi: 10.1371/journal.pone.0063074.
            Print 2013.
  PUBMED    23667577
REFERENCE   2  (bases 1 to 5798)
  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: "PLoS One.";
            date: "2013-05-7"; volume: "8(5):e63074. doi"; pages: " 
            10.1371/journal.pone.0063074. Print 2013"
COMMENT     Sequence Label: pCS2TAL3-RRR
FEATURES             Location/Qualifiers
     source          1..5798
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      85..94
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             97..117
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             118..207
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     CDS             complement(520..927)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG
                     TRQCIARCMHCLHCGHAATFCNLHKLACVQL"
     primer_bind     777..799
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     791..808
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     792..808
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        818..836
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(884..902)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(923..939)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    947..963
                     /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(971..977,978..995,996..1001))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1016..1037
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             1223..1810
                     /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
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQAREMQR
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNRITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     polyA_signal    1843..1977
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1867..1886)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1921..1940
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(2094..2112)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2133..2149)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2157..2173
                     /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(2181..2187,2188..2205,2206..2211))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2226..2247
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2364..2381)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2535..3123)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2615..2634)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3294..4085,4086..4154))
                     /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     3917..3936
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4155..4259)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4285..4740)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4422..4443)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4634..4653
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4814..5798
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atctgccacc atggctccaa agaagaagcg taaggtatac
      121 ccatacgatg ttcctgacta tgcgggctat ccctatgacg tcccggacta tgcaggatcg
      181 tatccatatg acgttccaga ttacgctgct catggtaccg tggatctacg cacgctcggc
      241 tacagtcagc agcagcaaga gaagatcaaa ccgaaggtgc gttcgacagt ggcgcagcac
      301 cacgaggcac tggtgggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac
      361 ccggcagcgt tagggaccgt cgctgtcacg tatcagcaca taatcacggc gttgccagag
      421 gcgacacacg aagacatcgt tggcgtcggc aaacagtggt ccggcgcacg cgccctggag
      481 gccttgctca cggatgcggg ggagttgaga ggtccgccgt tacagttgga cacaggccaa
      541 cttgtgaaga ttgcaaaacg tggcggcgtg accgcaatgg aggcagtgca tgcatcgcgc
      601 aatgcactga cgggtgcccc cctggagacg ggcgccgcta cagggcgcgt cccattcgcc
      661 attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca
      721 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
      781 gtcacgacgt tgtaaaacga cggccagtga gcgcgcgtaa tacgactcac tatagggcga
      841 attgggtacc gggccccccc tcgaggtcct ccagcttttg ttccctttag tgagggttaa
      901 ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
      961 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     1021 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccaccgg tcgtctccaa
     1081 cgaccacctc gtcgccttgg cctgcctcgg cggacgtcct gccatggatg cagtgaaaaa
     1141 gggattgccg cacgcgccgg aattgatcag aagagtcaat cgccgtattg gcgaacgcac
     1201 gtcccatcgc gttgccggat cccaactagt caaaagtgaa ctggaggaga agaaatctga
     1261 acttcgtcat aaattgaaat atgtgcctca tgaatatatt gaattaattg aaattgccag
     1321 aaattccact caggatagaa ttcttgaaat gaaggtaatg gaatttttta tgaaagttta
     1381 tggatataga ggtaaacatt tgggtggatc aaggaaaccg gacggagcaa tttatactgt
     1441 cggatctcct attgattacg gtgtgatcgt ggatactaaa gcttatagcg gaggttataa
     1501 tctgccaatt ggccaagcac gtgaaatgca acgatatgtc gaagaaaatc aaacacgaaa
     1561 caaacatatc aaccctaatg aatggtggaa agtctatcca tcttctgtaa cggaatttaa
     1621 gtttttattt gtgagtggtc actttaaagg aaactacaaa gctcagctta cacgattaaa
     1681 tagaatcact aattgtaatg gagctgttct tagtgtagaa gagcttttaa ttggtggaga
     1741 aatgattaaa gccggcacat taaccttaga ggaagtgcgc cggaaattta ataacggcga
     1801 gataaacttt taatctagaa ctatagtgag tcgtattacg tagatccaga catgataaga
     1861 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     1921 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     1981 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
     2041 ttcgcggccg cggcgccaat gcattgggcc cggtacgtac ccagcttttg ttccctttag
     2101 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     2161 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     2221 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     2281 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     2341 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     2401 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     2461 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     2521 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     2581 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     2641 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     2701 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     2761 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     2821 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     2881 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     2941 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     3001 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     3061 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     3121 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     3181 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     3241 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     3301 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     3361 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     3421 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     3481 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     3541 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3601 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     3661 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3721 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3781 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     3841 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3901 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3961 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     4021 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     4081 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     4141 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     4201 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     4261 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
     4321 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     4381 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     4441 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     4501 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     4561 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     4621 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     4681 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     4741 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     4801 tattacgcca gtcgaccata gccaattcaa tatggcgtat atggactcat gccaattcaa
     4861 tatggtggat ctggacctgt gccaattcaa tatggcgtat atggactcgt gccaattcaa
     4921 tatggtggat ctggacccca gccaattcaa tatggcggac ttggcaccat gccaattcaa
     4981 tatggcggac ttggcactgt gccaactggg gaggggtcta cttggcacgg tgccaagttt
     5041 gaggaggggt cttggccctg tgccaagtcc gccatattga attggcatgg tgccaataat
     5101 ggcggccata ttggctatat gccaggatca atatataggc aatatccaat atggccctat
     5161 gccaatatgg ctattggcca ggttcaatac tatgtattgg ccctatgcca tatagtattc
     5221 catatatggg ttttcctatt gacgtagata gcccctccca atgggcggtc ccatatacca
     5281 tatatggggc ttcctaatac cgcccatagc cactccccca ttgacgtcaa tggtctctat
     5341 atatggtctt tcctattgac gtcatatggg cggtcctatt gacgtatatg gcgcctcccc
     5401 cattgacgtc aattacggta aatggcccgc ctggctcaat gcccattgac gtcaatagga
     5461 ccacccacca ttgacgtcaa tgggatggct cattgcccat tcatatccgt tctcacgccc
     5521 cctattgacg tcaatgacgg taaatggccc acttggcagt acatcaatat ctattaatag
     5581 taacttggca agtacattac tattggaagg acgccagggt acattggcag tactcccatt
     5641 gacgtcaatg gcggtaaatg gcccgcgatg gctgccaagt acatccccat tgacgtcaat
     5701 ggggaggggc aatgacgcaa atgggcgttc cattgacgta aatgggcggt aggcgtgcct
     5761 aatgggaggt ctatataagc aatgctcgtt tagggaac
//
