LOCUS       sequence-418447-        4253 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate LI D-E module.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4253)
  AUTHORS   Tan H, Zhou Y, Dinius E, Lozano-Duran R
  TITLE     The Ti-TAN plasmid toolbox for TurboID-based proximity labeling 
            assays in Nicotiana benthamiana.
  JOURNAL   J Integr Plant Biol. 2024 Jan 31. doi: 10.1111/jipb.13610.
  PUBMED    38294100
REFERENCE   2  (bases 1 to 4253)
  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 Integr 
            Plant Biol. 2024 Jan 31. doi: 10.1111/jipb.13610."
COMMENT     Sequence Label: LI D-E TurboID-GFP
FEATURES             Location/Qualifiers
     source          1..4253
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     466..484
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(522..544)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     644..663
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     857..879
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     871..888
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     872..888
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             950..976
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             980..1006
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1010..1036
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1043..1999
                     /codon_start=1
                     /product="mutant of the E. coli biotin ligase BioID 
                     engineered for improved catalytic efficiency (Branon et 
                     al., 2018)"
                     /label=TurboID
                     /translation="KDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDW
                     GVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGD
                     ACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLG
                     ADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWI
                     TLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDK
                     EIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     CDS             join(2000..2002,2003..2716)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2303..2322)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2650..2671
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2777..2793)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2777..2793)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2790..2812)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2801..2817
                     /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(2825..2831,2832..2849,2850..2855))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2870..2891
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3008..3025)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3179..3767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3259..3278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(3882..3887,3888..3904,3905..3910)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             join(4099..4253,1..379)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
ORIGIN
        1 cgacgtagcc acctactccc aacatcagcc ggactccgat tacctcggga acttgctccg
       61 tagtaagaca ttcatcgcgc ttgctgcctt cgaccaagaa gcggttgttg gcgctctcgc
      121 ggcttacgtt ctgcccaagt ttgagcagcc gcgtagtgag atctatatct atgatctcgc
      181 agtctccgga gagcaccgga ggcagggcat tgccaccgcg ctcatcaatc tcctcaagca
      241 tgaggccaac gcgcttggtg cttatgtgat ctacgtgcaa gcagattacg gtgacgatcc
      301 cgcagtggct ctctatacaa agttgggcat acgggaagaa gtgatgcact ttgatatcga
      361 cccaagtacc gccacctaac aattcgttca agccgagatc ggcttcccgg ccgcggagtt
      421 gttcggtaaa ttgtcacaac gccgctcatg acattaacct ataaaaatag gcgtatcacg
      481 aggccctttt gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
      541 ccggagaagg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
      601 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
      661 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
      721 cgcatcaggc gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
      781 gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
      841 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attcgagctc
      901 ggtacctcgc gaatgcatct agatatcgga tccccgggtc tcaaaggcat acccgtatga
      961 tgttccggat tacgctggct atccctacga cgtgcccgac tatgccgggt acccctatga
     1021 cgtcccagac tacgcagcta gcaaagacaa tactgtgcct ctgaagctga tcgctctcct
     1081 ggctaatggc gagttccata gtggcgaaca gctgggagaa accctgggca tgtccagggc
     1141 cgctatcaac aagcacattc agactctgcg cgactggggc gtggacgtgt tcaccgtgcc
     1201 cggaaagggc tactctctgc ccgagcctat cccgctgctg aacgctaaac agattctggg
     1261 acagctggac ggcgggagcg tggcagtcct gcctgtggta gactccacca atcagtacct
     1321 gctggatcga atcggcgagc tgaagagtgg ggatgcttgc attgcagaat atcagcaggc
     1381 agggagagga agcagaggga ggaaatggtt ctctcctttt ggagctaacc tgtacctgag
     1441 tatgttttgg cgcctgaagc ggggaccagc agcaatcggc ctgggcccgg tcatcggaat
     1501 tgtcatggca gaagcgctgc gaaagctggg agcagacaag gtgcgagtca aatggcccaa
     1561 tgacctgtat ctgcaggata gaaagctggc aggcatcctg gtggagctgg ccggaataac
     1621 aggcgatgct gcacagatcg tcattggcgc cgggattaac gtggctatga ggcgcgtgga
     1681 ggaaagcgtg gtcaatcagg gctggatcac actgcaggaa gcagggatta acctggacag
     1741 gaatactctg gccgctacgc tgatccgaga gctgcgggca gccctggaac tgttcgagca
     1801 ggaaggcctg gctccatatc tgccacggtg ggagaagctg gataacttca tcaatagacc
     1861 cgtgaagctg atcattgggg acaaagagat tttcgggatt agccggggga ttgataaaca
     1921 gggagccctg ctgctggaac aggacggagt tatcaaaccc tggatgggcg gagaaatcag
     1981 tctgcggtct gccgaaaaga tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     2041 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     2101 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     2161 cgtgccctgg cccaccctcg tgaccacctt cacctacggc gtgcagtgct tcagccgcta
     2221 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     2281 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     2341 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     2401 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     2461 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     2521 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     2581 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     2641 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc acggcatgga
     2701 cgagctgtac aagtaaaatc tgagacctcg ggcccgtcga ctgcagaggc ctgcatgcaa
     2761 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     2821 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     2881 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     2941 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3001 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3061 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3121 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3181 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3241 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3301 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3361 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3421 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3481 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3541 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3601 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3661 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3721 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3781 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3841 tgaggcgcac accgtggaaa cggatgaagg cacgaaccca gttgacataa gcctgttcgg
     3901 ttcgtaaact gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga accttgaccg
     3961 aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttgttatgac tgtttttttg
     4021 tacagtctat gcctcgggca tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga
     4081 tgttatggag cagcaacgat gttacgcagc agcaacgatg ttacgcagca gggcagtcgc
     4141 cctaaaacaa agttaggtgg ctcaagtatg ggcatcattc gcacatgtag gctcggccct
     4201 gaccaagtca aatccatgcg ggctgctctt gatcttttcg gtcgtgagtt cgg
//
