LOCUS       sequence-246309-        4401 bp    DNA     circular SYN 14-APR-2025
DEFINITION  gateway entry vector for making N-terminal YFP-TurboID fusions with 
            non-nuclear proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4401)
  AUTHORS   Mair A, Xu SL, Branon TC, Ting AY, Bergmann DC
  TITLE     Proximity labeling of protein complexes and cell type-specific 
            organellar proteomes in Arabidopsis enabled by TurboID.
  JOURNAL   Elife. 2019 Sep 19;8. pii: 47864. doi: 10.7554/eLife.47864.
  PUBMED    31535972
REFERENCE   2  (bases 1 to 4401)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Elife."; 
            date: "2019-09-19"; volume: "8. pii: 47864. doi"; pages: " 
            10.7554/eLife.47864"
COMMENT     Sequence Label: pENTR_L1-YFP-Turbo-NES-L2
FEATURES             Location/Qualifiers
     source          1..4401
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     49..71
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     63..80
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     64..80
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    96..195
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(216..218,219..221,222..932)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(522..541)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     869..890
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             978..1019
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1026..1982
                     /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             1989..2015
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     protein_bind    complement(2053..2152)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2169..2188)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2170..2188)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2193..2209)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2193..2209)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2311..2330)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2322..3131
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2389..2408)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3278..3866
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3767..3786
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4020..4037
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(4196..4223)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4315..4401)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 atgcctggca gttccctact ctcgcgttaa cgctagcatg gatgttttcc cagtcacgac
       61 gttgtaaaac gacggccagt cttaagctcg ggccccaaat aatgatttta ttttgactga
      121 tagtgacctg ttcgttgcaa caaattgatg agcaatgctt ttttataatg ccaactttgt
      181 acaaaaaagc aggctccgcg gccgccccct tcaccatggt gagcaagggc gaggagctgt
      241 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
      301 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
      361 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccttc ggctacggcc
      421 tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
      481 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
      541 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
      601 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
      661 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
      721 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
      781 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagctaccag tccgccctga
      841 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
      901 ggatcactct cggcatggac gagctgtaca aggctaaggg tgggcgcgcc ggtggaggcg
      961 gttcaggagg tggcatgggc aagcccatcc ccaaccccct gctgggcctg gacagcaccg
     1021 ctagcaaaga caatactgtg cctctgaagc tgatcgctct cctggctaat ggcgagttcc
     1081 atagtggcga acagctggga gaaaccctgg gcatgtccag ggccgctatc aacaagcaca
     1141 ttcagactct gcgcgactgg ggcgtggacg tgttcaccgt gcccggaaag ggctactctc
     1201 tgcccgagcc tatcccgctg ctgaacgcta aacagattct gggacagctg gacggcggga
     1261 gcgtggcagt cctgcctgtg gtcgactcca ccaatcagta cctgctggat cgaatcggcg
     1321 agctgaagag tggggatgct tgcattgcag aatatcagca ggcagggaga ggaagcagag
     1381 ggaggaaatg gttctctcct tttggagcta acctgtacct gagtatgttt tggcgcctga
     1441 agcggggacc agcagcaatc ggcctgggcc cggtcatcgg aattgtcatg gcagaagcgc
     1501 tgcgaaagct gggagcagac aaggtgcgag tcaaatggcc caatgacctg tatctgcagg
     1561 atagaaagct ggcaggcatc ctggtggagc tggccggaat aacaggcgat gctgcacaga
     1621 tcgtcattgg cgccgggatt aacgtggcta tgaggcgcgt ggaggaaagc gtggtcaatc
     1681 agggctggat cacactgcag gaagcaggga ttaacctgga caggaatact ctggccgcta
     1741 cgctgatccg agagctgcgg gcagccctgg aactgttcga gcaggaaggc ctggctccat
     1801 atctgccacg gtgggagaag ctggataact tcatcaatag acccgtgaag ctgatcattg
     1861 gggacaaaga gattttcggg attagccggg ggattgataa acagggagcc ctgctgctgg
     1921 aacaggacgg agttatcaaa ccctggatgg gcggagaaat cagtctgcgg tctgccgaaa
     1981 agctgcagct gcctcccctg gagcgcctga ccctggacgg aggcggtgga agcggcggag
     2041 gtgggcgcgc cgacccagct ttcttgtaca aagttggcat tataagaaag cattgcttat
     2101 caatttgttg caacgaacag gtcactatca gtcaaaataa aatcattatt tgccatccag
     2161 ctgatatccc ctatagtgag tcgtattaca tggtcatagc tgtttcctgg cagctctggc
     2221 ccgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa
     2281 taaaactgtc tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg
     2341 aaacgtcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg
     2401 ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg
     2461 cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga
     2521 tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc
     2581 gtactcctga tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg
     2641 tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc
     2701 gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc
     2761 tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg
     2821 agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct
     2881 caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg
     2941 ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc
     3001 ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc
     3061 aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg
     3121 agtttttcta atcagaattg gttaattggt tgtaacactg gcagagcatt acgctgactt
     3181 gacgggacgg cgcaagctca tgaccaaaat cccttaacgt gagttacgcg tcgttccact
     3241 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3301 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3361 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3421 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3481 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3541 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3601 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3661 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3721 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3781 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3841 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3901 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3961 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4021 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     4081 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     4141 agcgcaacgc aattaatacg cgtaccgcta gccaggaaga gtttgtagaa acgcaaaaag
     4201 gccatccgtc aggatggcct tctgcttagt ttgatgcctg gcagtttatg gcgggcgtcc
     4261 tgcccgccac cctccgggcc gttgcttcac aacgttcaaa tccgctcccg gcggatttgt
     4321 cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtcttccga
     4381 ctgagccttt cgttttattt g
//
