LOCUS       sequence-78693-e        4659 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway entry vector with attL1 and attR5 sites. Between the att 
            sites are two FokI coding sequences transcriptionally linked by a 
            T2A sequence. There is a nos-T after the last FokI..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4659)
  AUTHORS   Baltes NJ, Gil-Humanes J, Cermak T, Atkins PA, Voytas DF
  TITLE     DNA Replicons for Plant Genome Engineering.
  JOURNAL   Plant Cell. 2014 Jan;26(1):151-63. doi: 10.1105/tpc.113.119792
  PUBMED    24443519
REFERENCE   2  (bases 1 to 4659)
  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"; doi: "10.1105/tpc.113"; 
            journalName: "Plant Cell"; date: "2014-01"; volume: "26"; issue: 
            "1"; pages: "151-63"
COMMENT     Sequence Label: pNJB91
FEATURES             Location/Qualifiers
     source          1..4659
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      8..260
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    305..428
                     /gene="mutant version of attR"
                     /label=attR5
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(445..464)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(446..464)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(469..485)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(469..485)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             916..1707
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      1800..2388
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2289..2308
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2542..2559
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2718..2745)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2837..2923)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2972..2994
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2986..3003
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2987..3003
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3019..3118
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3140..3205
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3212..3232
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3873..3926
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3933..3998
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4005..4025
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4066..4653
                     /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
                     MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMER
                     YVEENQTRNKHLNPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL
                     SVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF"
ORIGIN
        1 gacgtccgat cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg
       61 tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat
      121 gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat
      181 ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt
      241 gtcatctatg ttactagatc gggaattgat cccccctcga cagcttccgg aaagggcgaa
      301 ttcgcaactt tgtatacaaa agttgaacga gaaacgtaaa atgatataaa tatcaatata
      361 ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca acatatccag
      421 tcactatgcc atccagctga tatcccctat agtgagtcgt attacatggt catagctgtt
      481 tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat
      541 atatcatcat gcctcctctg gaccagccag gacagaaatg cctcgacttc gctgctaccc
      601 aaggttgccg ggtgacgcac accgtggaaa cggatgaagg cacgaaccca gtggacataa
      661 gcctgttcgg ttcgtaagct gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga
      721 accttgaccg aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttgttatgac
      781 tgtttttttg gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg
      841 ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa
      901 acaaagttaa acatcatgag ggaagcggtg atcgccgaag tatcgactca actatcagag
      961 gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc
     1021 tccgcagtgg atggcggcct gaagccacac agtgatattg atttgctggt tacggtgacc
     1081 gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct
     1141 tcccctggag agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac
     1201 atcattccgt ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat
     1261 gacattcttg caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg
     1321 acaaaagcaa gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat
     1381 ccggttcctg aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg
     1441 ccgcccgact gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac
     1501 agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc
     1561 ctgccggccc agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa
     1621 gaagatcgct tggcctcgcg cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc
     1681 gagatcacca aggtagtcgg caaataaccc tcgagccacc catgaccaaa atcccttaac
     1741 gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     1801 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     1861 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     1921 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     1981 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2041 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     2101 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     2161 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     2221 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     2281 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2341 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     2401 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     2461 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     2521 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     2581 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     2641 cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc gagccaggaa
     2701 gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta gtttgatgcc
     2761 tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc acaacgttca
     2821 aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa
     2881 aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg gcagttccct
     2941 actctcgcgt taacgcttgc atggatgttt tcccagtcac gacgttgtaa aacgacggcc
     3001 agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac ctgttcgttg
     3061 caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa agcaggctcc
     3121 gaattcgccc ttcaccatgg attataagga tcacgatggc gactacaagg accacgatat
     3181 tgactacaaa gacgatgacg ataaaatggc tcctaagaaa aagcgcaaag tcggtatcca
     3241 tggcgttccc tctagataac gcaggatccc agctggtgaa gtccgagctg gaagaaaaaa
     3301 agagcgagct gcgccacaag ctcaagtacg tgccccacga gtacatcgag ctgatcgaga
     3361 tcgcccgcaa cagcacccaa gaccgcatcc tggagatgaa agtgatggag ttcttcatga
     3421 aggtgtacgg ctaccgcggc aagcacctgg gcggctcccg caagcccgat ggcgccatct
     3481 acaccgtggg ctcccccatc gactatggcg tcattgtcga caccaaggcc tactccggcg
     3541 gctacaactt acccatcggt caggccgacg agatgcaacg ctacgtgaag gagaaccaga
     3601 cccgcaataa gcacattaat cccaacgagt ggtggaaggt gtacccctcc tccgtgaccg
     3661 agttcaaatt cctgttcgtg tccggccact tcaagggcaa ttataaggcc caactgaccc
     3721 gcctgaacca caagaccaac tgcaacggcg ccgtgctgtc cgtggaggaa ctgctgatcg
     3781 gcggcgagat gatcaaggct ggtaccctga ccctggaaga ggtgcgccgc aagttcaaca
     3841 atggtgaaat caatttcagg tccggcggcg gagagggcag aggaagtctt ctaacatgcg
     3901 gtgacgtgga ggagaatccc ggccctagga tggactacaa agaccatgac ggtgattata
     3961 aagatcatga catcgattac aaggatgacg atgacaagat ggcccccaag aagaagagga
     4021 aggtgggcat tcacggggtg ccggctagct agtaagctta gatctcaact agtcaaaagt
     4081 gaactggagg agaagaaatc tgaacttcgt cataaattga aatatgtgcc tcatgaatat
     4141 attgaattaa ttgaaattgc cagaaattcc actcaggata gaattcttga aatgaaggta
     4201 atggaatttt ttatgaaagt ttatggatat agaggtaaac atttgggtgg atcaaggaaa
     4261 ccggacggag caatttatac tgtcggatct cctattgatt acggtgtgat cgtggatact
     4321 aaagcttata gcggaggtta taatctgcca attggccaag cagatgaaat ggagcgatat
     4381 gtcgaagaaa atcaaacacg aaacaaacat ctcaacccta atgaatggtg gaaagtctat
     4441 ccatcttctg taacggaatt taagttttta tttgtgagtg gtcactttaa aggaaactac
     4501 aaagctcagc ttacacgatt aaatcatatc actaattgta atggagctgt tcttagtgta
     4561 gaagagcttt taattggtgg agaaatgatt aaagccggca cattaacctt agaggaagtg
     4621 agacggaaat ttaataacgg cgagataaac ttttaatag
//
