LOCUS       sequence-51019-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 heterodimeric FokI domain
            (KKR)..
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)-FokI-KKR-Destination
FEATURES             Location/Qualifiers
     source          1..6441
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    19..40
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(55..60,61..78,79..85)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    93..109
                     /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     117..133
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        153..532
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..811
                     /label=chicken beta-actin promoter
     intron          812..1820
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1744..1766
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        1833..1851
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1871..1891
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(2195..2602)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG
                     TRQCIARCMHCLHCGHAATFCNLHKLACVQL"
     primer_bind     2467..2483
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2493..2511
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2559..2579)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2559..2577)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2598..2614)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2622..2638
                     /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(2646..2652,2653..2670,2671..2676))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2691..2712
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3507..3524)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3513..3737
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3809..3825)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      3967..4422
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4054..4073)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4264..4285
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4448..4552
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4553..4621,4622..5413)
                     /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(4771..4790)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5584..6172
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6073..6092
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6326..6343
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
       61 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
      121 aaacagctat gaccatgagg cgcgccggat tcgacattga ttattgacta gttattaata
      181 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
      241 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
      301 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
      361 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
      421 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
      481 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtcgaggt
      541 gagccccacg ttctgcttca ctctccccat ctcccccccc tccccacccc caattttgta
      601 tttatttatt ttttaattat tttgtgcagc gatgggggcg gggggggggg gggggcgcgc
      661 gccaggcggg gcggggcggg gcgaggggcg gggcggggcg aggcggagag gtgcggcggc
      721 agccaatcag agcggcgcgc tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg
      781 gccctataaa aagcgaagcg cgcggcgggc gggagtcgct gcgttgcctt cgccccgtgc
      841 cccgctccgc gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca
      901 ggtgagcggg cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg
      961 gctcgtttct tttctgtggc tgcgtgaaag ccttaaaggg ctccgggagg gccctttgtg
     1021 cgggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg
     1081 gcccgcgctg cccggcggct gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg
     1141 cgtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg ggctgcgagg
     1201 ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgg
     1261 cggtcgggct gtaacccccc cctgcacccc cctccccgag ttgctgagca cggcccggct
     1321 tcgggtgcgg ggctccgtgc ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg
     1381 cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg ccggggaggg ctcgggggag
     1441 gggcgcggcg gccccggagc gccggcggct gtcgaggcgc ggcgagccgc agccattgcc
     1501 ttttatggta atcgtgcgag agggcgcagg gacttccttt gtcccaaatc tggcggagcc
     1561 gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc gggcgaagcg gtgcggcgcc
     1621 ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc
     1681 catctccagc ctcggggctg ccgcaggggg acggctgcct tcggggggga cggggcaggg
     1741 cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg
     1801 ccttcttctt tttcctacag atccttaatt aataatacga ctcactatag gggccgccac
     1861 catgagatct cctaagaaaa agaggaaggt gcaggtggat ctacgcacgc tcggctacag
     1921 tcagcagcag caagagaaga tcaaaccgaa ggtgcgttcg acagtggcgc agcaccacga
     1981 ggcactggtg ggccatgggt ttacacacgc gcacatcgtt gcgctcagcc aacacccggc
     2041 agcgttaggg accgtcgctg tcacgtatca gcacataatc acggcgttgc cagaggcgac
     2101 acacgaagac atcgttggcg tcggcaaaca gtggtccggc gcacgcgccc tggaggcctt
     2161 gctcacggat gcgggggagt tgagaggtcc gccgttacag ttggacacag gccaacttgt
     2221 gaagattgca aaacgtggcg gcgtgaccgc aatggaggca gtgcatgcat cgcgcaatgc
     2281 actgacgggt gcccccctgg agacgggcgc cgctacaggg cgcgtcccat tcgccattca
     2341 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
     2401 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
     2461 gacgttgtaa aacgacggcc agtgagcgcg cgtaatacga ctcactatag ggcgaattgg
     2521 gtaccgggcc ccccctcgag gtcctccagc ttttgttccc tttagtgagg gttaattgcg
     2581 cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     2641 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     2701 taactcacat taattgcgtt gcgctcactg cccgctttcc accggtcgtc tccaacgacc
     2761 acctcgtcgc cttggcctgc ctcggcggac gtcctgccat ggatgcagtg aaaaagggat
     2821 tgccgcacgc gccggaattg atcagaagag tcaatcgccg tattggcgaa cgcacgtccc
     2881 atcgcgttgc gggatcccag ctggtgaaga gcgagctgga ggagaagaag tccgagctgc
     2941 ggcacaagct gaagtacgtg ccccacgagt acatcgagct gatcgagatc gccaggaacc
     3001 ccacccagga ccgcatcctg gagatgaagg tgatggagtt cttcatgaag gtgtacggct
     3061 acaggggaga gcacctgggc ggaagcagaa agcctgacgg cgccatctat acagtgggca
     3121 gccccatcga ttacggcgtg atcgtggaca caaaggccta cagcggcggc tacaatctgc
     3181 ctatcggcca ggccgacgag atgcagagat acgtgaagga gaaccagacc cggaataagc
     3241 acatcaaccc caacgagtgg tggaaggtgt accctagcag cgtgaccgag ttcaagttcc
     3301 tgttcgtgag cggccacttc aagggcaact acaaggccca gctgaccagg ctgaaccgca
     3361 aaaccaactg caatggcgcc gtgctgagcg tggaggagct gctgatcggc ggcgagatga
     3421 tcaaagccgg caccctgaca ctggaggagg tgcggcgcaa gttcaacaac ggcgagatca
     3481 acttctgata ggagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     3541 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     3601 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     3661 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     3721 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga gatccactag
     3781 ttctagcctc gaggctagag cggccgccac tggccgtcgt tttacaacgt cgtgactggg
     3841 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     3901 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     3961 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4021 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     4081 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4141 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     4201 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4261 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     4321 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     4381 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg
     4441 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     4501 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     4561 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     4621 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     4681 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     4741 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     4801 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     4861 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     4921 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     4981 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     5041 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     5101 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     5161 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     5221 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     5281 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     5341 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     5401 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     5461 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     5521 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     5581 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     5641 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     5701 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     5761 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     5821 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     5881 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     5941 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     6001 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     6061 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     6121 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     6181 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     6241 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     6301 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     6361 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     6421 gtttcccgac tggaaagcgg g
//
