LOCUS       Exported                5768 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  TALEN backbone vector for use with the Golden Gate TALEN Kit in 
            place of pTAL1-4. Shorter N- and C-terminal tal protein segments 
            increases mutation induction; Fok I DD (improved 
            version=pCS2TAL3-DDD).
ACCESSION   .
VERSION     .
KEYWORDS    pCS2TAL3-DD
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5768)
  AUTHORS   Dahlem TJ, Hoshijima K, Jurynec MJ, Gunther D, Starker CG, Locke AS,
            Weis AM, Voytas DF, Grunwald DJ
  TITLE     Simple Methods for Generating and Detecting Locus-Specific Mutations
            Induced with TALENs in the Zebrafish Genome.
  JOURNAL   PLoS Genet. 2012 Aug;8(8):e1002861. Epub 2012 Aug 16.
  PUBMED    22916025
REFERENCE   2  (bases 1 to 5768)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5768
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     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..183
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             complement(490..897)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG
                     TRQCIARCMHCLHCGHAATFCNLHKLACVQL"
     primer_bind     747..769
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     761..778
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     762..778
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     788..807
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        788..806
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(854..872)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(893..909)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    917..933
                     /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(941..971)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    986..1007
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             1193..1780
                     /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
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQD
                     YVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
     promoter        complement(1791..1808)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    1813..1947
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1837..1856)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1891..1910
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(2064..2082)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2103..2119)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2127..2143
                     /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(2151..2181)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2196..2217
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2334..2351)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2505..3093)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2585..2604)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3264..4124)
                     /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     3887..3906
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4125..4229)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4255..4710)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4392..4413)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4604..4623
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4784..5768
                     /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 taaggtagac
      121 tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga tgacgatgac
      181 aagggtaccg tggatctacg cacgctcggc tacagtcagc agcagcaaga gaagatcaaa
      241 ccgaaggtgc gttcgacagt ggcgcagcac cacgaggcac tggtgggcca tgggtttaca
      301 cacgcgcaca tcgttgcgct cagccaacac ccggcagcgt tagggaccgt cgctgtcacg
      361 tatcagcaca taatcacggc gttgccagag gcgacacacg aagacatcgt tggcgtcggc
      421 aaacagtggt ccggcgcacg cgccctggag gccttgctca cggatgcggg ggagttgaga
      481 ggtccgccgt tacagttgga cacaggccaa cttgtgaaga ttgcaaaacg tggcggcgtg
      541 accgcaatgg aggcagtgca tgcatcgcgc aatgcactga cgggtgcccc cctggagacg
      601 ggcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg
      661 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg
      721 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
      781 gcgcgcgtaa tacgactcac tatagggcga attgggtacc gggccccccc tcgaggtcct
      841 ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc
      901 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
      961 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     1021 cactgcccgc tttccaccgg tcgtctccaa cgaccacctc gtcgccttgg cctgcctcgg
     1081 cggacgtcct gccatggatg cagtgaaaaa gggattgccg cacgcgccgg aattgatcag
     1141 aagagtcaat cgccgtattg gcgaacgcac gtcccatcgc gttgccggat cccaactagt
     1201 caaaagtgaa ctggaggaga agaaatctga acttcgtcat aaattgaaat atgtgcctca
     1261 tgaatatatt gaattaattg aaattgccag aaattccact caggatagaa ttcttgaaat
     1321 gaaggtaatg gaatttttta tgaaagttta tggatataga ggtaaacatt tgggtggatc
     1381 aaggaaaccg gacggagcaa tttatactgt cggatctcct attgattacg gtgtgatcgt
     1441 ggatactaaa gcttatagcg gaggttataa tctgccaatt ggccaagcag atgaaatgca
     1501 agactatgtc gaagaaaatc aaacacgaaa caaacatatc aaccctaatg aatggtggaa
     1561 agtctatcca tcttctgtaa cggaatttaa gtttttattt gtgagtggtc actttaaagg
     1621 aaactacaaa gctcagctta cacgattaaa tcatatcact aattgtaatg gagctgttct
     1681 tagtgtagaa gagcttttaa ttggtggaga aatgattaaa gccggcacat taaccttaga
     1741 ggaagtgcgc cggaaattta ataacggcga gataaacttt taatctagaa ctatagtgag
     1801 tcgtattacg tagatccaga catgataaga tacattgatg agtttggaca aaccacaact
     1861 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     1921 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     1981 gttcaggggg aggtgtggga ggttttttaa ttcgcggccg cggcgccaat gcattgggcc
     2041 cggtacgtac ccagcttttg ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca
     2101 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     2161 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     2221 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     2281 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     2341 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     2401 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     2461 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2521 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2581 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2641 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2701 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2761 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2821 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2881 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     2941 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3001 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3061 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3121 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3181 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3241 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3301 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3361 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     3421 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     3481 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3541 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3601 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3661 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3721 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3781 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3841 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3901 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     3961 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4021 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4081 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4141 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4201 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg
     4261 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     4321 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     4381 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     4441 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     4501 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     4561 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     4621 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     4681 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt
     4741 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gtcgaccata gccaattcaa
     4801 tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt gccaattcaa
     4861 tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca gccaattcaa
     4921 tatggcggac ttggcaccat gccaattcaa tatggcggac ttggcactgt gccaactggg
     4981 gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg tgccaagtcc
     5041 gccatattga attggcatgg tgccaataat ggcggccata ttggctatat gccaggatca
     5101 atatataggc aatatccaat atggccctat gccaatatgg ctattggcca ggttcaatac
     5161 tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt gacgtagata
     5221 gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac cgcccatagc
     5281 cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac gtcatatggg
     5341 cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta aatggcccgc
     5401 ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa tgggatggct
     5461 cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg taaatggccc
     5521 acttggcagt acatcaatat ctattaatag taacttggca agtacattac tattggaagg
     5581 acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg gcccgcgatg
     5641 gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa atgggcgttc
     5701 cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc aatgctcgtt
     5761 tagggaac
//
