LOCUS       sequence-51020-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
            (ELD)..
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-ELD-Destination
FEATURES             Location/Qualifiers
     source          1..6441
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(12..17,18..35,36..42)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    50..66
                     /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     74..90
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        110..489
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        491..768
                     /label=chicken beta-actin promoter
     intron          769..1777
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1701..1723
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        1790..1808
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1828..1848
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(2152..2559)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGARLQGG
                     TRQCIARCMHCLHCGHAATFCNLHKLACVQL"
     primer_bind     2424..2440
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2450..2468
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2516..2536)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2516..2534)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2555..2571)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2579..2595
                     /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(2603..2609,2610..2627,2628..2633))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2648..2669
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3464..3481)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3470..3694
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3766..3782)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      3924..4379
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4011..4030)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4221..4242
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4405..4509
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4510..4578,4579..5370)
                     /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(4728..4747)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5541..6129
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6030..6049
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6283..6300
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6417..6438
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
       61 taacaatttc acacaggaaa cagctatgac catgaggcgc gccggattcg acattgatta
      121 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      181 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      241 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      301 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      361 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      421 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      481 attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc
      541 ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg
      601 gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg
      661 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
      721 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
      781 ttgccttcgc cccgtgcccc gctccgcgcc gcctcgcgcc gcccgccccg gctctgactg
      841 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
      901 cgcttggttt aatgacggct cgtttctttt ctgtggctgc gtgaaagcct taaagggctc
      961 cgggagggcc ctttgtgcgg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt
     1021 ggggagcgcc gcgtgcggcc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg
     1081 gggctttgtg cgctccgcgt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt
     1141 gcgggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc
     1201 agggggtgtg ggcgcggcgg tcgggctgta acccccccct gcacccccct ccccgagttg
     1261 ctgagcacgg cccggcttcg ggtgcggggc tccgtgcggg gcgtggcgcg gggctcgccg
     1321 tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg
     1381 gggagggctc gggggagggg cgcggcggcc ccggagcgcc ggcggctgtc gaggcgcggc
     1441 gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc
     1501 ccaaatctgg cggagccgaa atctgggagg cgccgccgca ccccctctag cgggcgcggg
     1561 cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc
     1621 gccgccgtcc ccttctccat ctccagcctc ggggctgccg cagggggacg gctgccttcg
     1681 ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct ctagagcctc
     1741 tgctaaccat gttcatgcct tcttcttttt cctacagatc cttaattaat aatacgactc
     1801 actatagggg ccgccaccat gagatctcct aagaaaaaga ggaaggtgca ggtggatcta
     1861 cgcacgctcg gctacagtca gcagcagcaa gagaagatca aaccgaaggt gcgttcgaca
     1921 gtggcgcagc accacgaggc actggtgggc catgggttta cacacgcgca catcgttgcg
     1981 ctcagccaac acccggcagc gttagggacc gtcgctgtca cgtatcagca cataatcacg
     2041 gcgttgccag aggcgacaca cgaagacatc gttggcgtcg gcaaacagtg gtccggcgca
     2101 cgcgccctgg aggccttgct cacggatgcg ggggagttga gaggtccgcc gttacagttg
     2161 gacacaggcc aacttgtgaa gattgcaaaa cgtggcggcg tgaccgcaat ggaggcagtg
     2221 catgcatcgc gcaatgcact gacgggtgcc cccctggaga cgggcgccgc tacagggcgc
     2281 gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     2341 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     2401 agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc
     2461 actatagggc gaattgggta ccgggccccc cctcgaggtc ctccagcttt tgttcccttt
     2521 agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     2581 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     2641 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccacc
     2701 ggtcgtctcc aacgaccacc tcgtcgcctt ggcctgcctc ggcggacgtc ctgccatgga
     2761 tgcagtgaaa aagggattgc cgcacgcgcc ggaattgatc agaagagtca atcgccgtat
     2821 tggcgaacgc acgtcccatc gcgttgcggg atcccagctg gtgaagagcg agctggagga
     2881 gaagaagtcc gagctgcggc acaagctgaa gtacgtgccc cacgagtaca tcgagctgat
     2941 cgagatcgcc aggaacccca cccaggaccg catcctggag atgaaggtga tggagttctt
     3001 catgaaggtg tacggctaca ggggagagca cctgggcgga agcagaaagc ctgacggcgc
     3061 catctataca gtgggcagcc ccatcgatta cggcgtgatc gtggacacaa aggcctacag
     3121 cggcggctac aatctgccta tcggccaggc cgacgagatg gagagatacg tggaggagaa
     3181 ccagacacgg gataagcacc tcaaccccaa cgagtggtgg aaggtgtacc ctagcagcgt
     3241 gaccgagttc aagttcctgt tcgtgagcgg ccacttcaag ggcaactaca aggcccagct
     3301 gaccaggctg aaccacatca ccaactgcaa tggcgccgtg ctgagcgtgg aggagctgct
     3361 gatcggcggc gagatgatca aagccggcac cctgacactg gaggaggtgc ggcgcaagtt
     3421 caacaacggc gagatcaact tctgatagga gctcgctgat cagcctcgac tgtgccttct
     3481 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     3541 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     3601 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     3661 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     3721 ggctcgagat ccactagttc tagcctcgag gctagagcgg ccgccactgg ccgtcgtttt
     3781 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     3841 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     3901 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     3961 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     4021 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     4081 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     4141 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     4201 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     4261 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     4321 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     4381 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     4441 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     4501 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     4561 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     4621 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     4681 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     4741 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     4801 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     4861 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     4921 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     4981 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5041 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     5101 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     5161 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     5221 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     5281 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     5341 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     5401 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     5461 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     5521 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     5581 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     5641 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     5701 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     5761 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     5821 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     5881 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     5941 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6001 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6061 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6121 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6181 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     6241 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     6301 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     6361 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     6421 gtgagttagc tcactcatta g
//
