LOCUS       sequence-46727-e        6441 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination vector for Voytas Golden Gate TALEN assembly 
            incorporating a CAG promoter for mammalian expression and a T7 
            promoter for synthesis of TALEN mRNA. Uses homodimeric FokI 
            domains..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6441)
  AUTHORS   Hermann M, Cermak T, Voytas DF, Pelczar P
  TITLE     Mouse genome engineering using designer nucleases.
  JOURNAL   J Vis Exp. 2014 Apr 2;(86). doi: 10.3791/50930.
  PUBMED    24747757
REFERENCE   2  (bases 1 to 6441)
  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: "J Vis 
            Exp."; date: "2014-04-2"; volume: "(86). doi"; pages: " 
            10.3791/50930"
COMMENT     Sequence Label: pCAG-T7-TALEN(Sangamo)-Destination
FEATURES             Location/Qualifiers
     source          1..6441
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    50..71
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(86..91,92..109,110..116)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    124..140
                     /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)."
     primer_bind     148..164
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        184..563
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        565..842
                     /label=chicken beta-actin promoter
     intron          843..1851
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1775..1797
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        1864..1882
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1902..1922
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(2226..2633)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG
                     TRQCIARCMHCLHCGHAATFCNLHKLACVQL"
     primer_bind     2498..2514
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2524..2542
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2590..2610)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2590..2608)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2629..2645)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2653..2669
                     /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(2677..2683,2684..2701,2702..2707))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2722..2743
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             2929..3516
                     /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"
     primer_bind     complement(3538..3555)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3544..3768
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3840..3856)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      3998..4453
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4085..4104)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4295..4316
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4479..4583
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4584..4652,4653..5444)
                     /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     complement(4802..4821)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5615..6203
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6104..6123
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6357..6374
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
       61 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
      121 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgag gcgcgccgga
      181 ttcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      241 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      301 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      361 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      421 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      481 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      541 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      601 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      661 cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc
      721 ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
      781 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
      841 cgggagtcgc tgcgttgcct tcgccccgtg ccccgctccg cgccgcctcg cgccgcccgc
      901 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
      961 cgggctgtaa ttagcgcttg gtttaatgac ggctcgtttc ttttctgtgg ctgcgtgaaa
     1021 gccttaaagg gctccgggag ggccctttgt gcggggggga gcggctcggg gggtgcgtgc
     1081 gtgtgtgtgt gcgtggggag cgccgcgtgc ggcccgcgct gcccggcggc tgtgagcgct
     1141 gcgggcgcgg cgcggggctt tgtgcgctcc gcgtgtgcgc gaggggagcg cggccggggg
     1201 cggtgccccg cggtgcgggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc
     1261 gtgggggggt gagcaggggg tgtgggcgcg gcggtcgggc tgtaaccccc ccctgcaccc
     1321 ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgtg cggggcgtgg
     1381 cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg
     1441 gccgcctcgg gccggggagg gctcggggga ggggcgcggc ggccccggag cgccggcggc
     1501 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1561 ggacttcctt tgtcccaaat ctggcggagc cgaaatctgg gaggcgccgc cgcaccccct
     1621 ctagcgggcg cgggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct
     1681 tcgtgcgtcg ccgcgccgcc gtccccttct ccatctccag cctcggggct gccgcagggg
     1741 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc
     1801 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gatccttaat
     1861 taataatacg actcactata ggggccgcca ccatgagatc tcctaagaaa aagaggaagg
     1921 tgcaggtgga tctacgcacg ctcggctaca gtcagcagca gcaagagaag atcaaaccga
     1981 aggtgcgttc gacagtggcg cagcaccacg aggcactggt gggccatggg tttacacacg
     2041 cgcacatcgt tgcgctcagc caacacccgg cagcgttagg gaccgtcgct gtcacgtatc
     2101 agcacataat cacggcgttg ccagaggcga cacacgaaga catcgttggc gtcggcaaac
     2161 agtggtccgg cgcacgcgcc ctggaggcct tgctcacgga tgcgggggag ttgagaggtc
     2221 cgccgttaca gttggacaca ggccaacttg tgaagattgc aaaacgtggc ggcgtgaccg
     2281 caatggaggc agtgcatgca tcgcgcaatg cactgacggg tgcccccctg gagacgggcg
     2341 ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg
     2401 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta
     2461 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc
     2521 gcgtaatacg actcactata gggcgaattg ggtaccgggc cccccctcga ggtcctccag
     2581 cttttgttcc ctttagtgag ggttaattgc gcgcttggcg taatcatggt catagctgtt
     2641 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     2701 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     2761 gcccgctttc caccggtcgt ctccaacgac cacctcgtcg ccttggcctg cctcggcgga
     2821 cgtcctgcca tggatgcagt gaaaaaggga ttgccgcacg cgccggaatt gatcagaaga
     2881 gtcaatcgcc gtattggcga acgcacgtcc catcgcgttg cgggatccca gctagtgaaa
     2941 tctgaattgg aagagaagaa atctgaactt agacataaat tgaaatatgt gccacatgaa
     3001 tatattgaat tgattgaaat cgcaagaaat tcaactcagg atagaatcct tgaaatgaag
     3061 gtgatggagt tctttatgaa ggtttatggt tatcgtggta aacatttggg tggatcaagg
     3121 aaaccagacg gagcaattta tactgtcgga tctcctattg attacggtgt gatcgttgat
     3181 actaaggcat attcaggagg ttataatctt ccaattggtc aagcagatga aatgcaaaga
     3241 tatgtcgaag agaatcaaac aagaaacaag catatcaacc ctaatgaatg gtggaaagtc
     3301 tatccatctt cagtaacaga atttaagttc ttgtttgtga gtggtcattt caaaggaaac
     3361 tacaaagctc agcttacaag attgaatcat atcactaatt gtaatggagc tgttcttagt
     3421 gtagaagagc ttttgattgg tggagaaatg attaaagctg gtacattgac acttgaggaa
     3481 gtgagaagga aatttaataa cggtgagata aacttttaat aggagctcgc tgatcagcct
     3541 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3601 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3661 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3721 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     3781 aaagaaccag ctggggctcg agatccacta gttctagcct cgaggctaga gcggccgcca
     3841 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     3901 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     3961 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta
     4021 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     4081 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     4141 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     4201 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     4261 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     4321 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     4381 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     4441 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     4501 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     4561 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     4621 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     4681 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     4741 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     4801 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     4861 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     4921 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     4981 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     5041 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     5101 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     5161 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     5221 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     5281 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     5341 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     5401 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     5461 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     5521 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     5581 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     5641 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     5701 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     5761 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     5821 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     5881 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     5941 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6001 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6061 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     6121 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     6181 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     6241 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     6301 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     6361 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     6421 ggccgattca ttaatgcagc t
//
