LOCUS       sequence-432140-        5713 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway shuttling vector for APEX2-tagged P4C, a P4C biosensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5713)
  AUTHORS   Hollingsworth LR, Veeraraghavan P, Paulo JA, Harper JW
  TITLE     Spatiotemporal proteomic profiling of cellular responses to NLRP3 
            agonists
  JOURNAL   bioRxiv 2024.04.19.590338
REFERENCE   2  (bases 1 to 5713)
  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: "bioRxiv 
            2024.04.19.590338"
COMMENT     Sequence Label: pDONR221 FLAG-APEX2-P4C-IRES-mNeonGreen
FEATURES             Location/Qualifiers
     source          1..5713
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    569..668
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             677..700
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             701..1447
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     CDS             1448..1471
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     misc_feature    2005..2557
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2149..2166)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2376..2395
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2569..3279
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      2824..2833
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3281..3380)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3397..3416)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3398..3416)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3421..3437)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3421..3437)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(4023..4838)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4023..4838)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      5063..5651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5552..5571
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac
      601 ctgttcgttg caacaaattg atgagcaatg cttttttata atgccaactt tgtacaaaaa
      661 agcaggctcc accatggact acaaggatga cgacgataag ggaaagtctt acccaactgt
      721 gagtgctgat taccaggacg ccgttgagaa ggcgaagaag aagctcagag gcttcatcgc
      781 tgagaagaga tgcgctcctc taatgctccg tttggcattc cactctgctg gaacctttga
      841 caagggcacg aagaccggtg gacccttcgg aaccatcaag caccctgccg aactggctca
      901 cagcgctaac aacggtcttg acatcgctgt taggcttttg gagccactca aggcggagtt
      961 ccctattttg agctacgccg atttctacca gttggctggc gttgttgccg ttgaggtcac
     1021 gggtggacct aaggttccat tccaccctgg aagagaggac aagcctgagc caccaccaga
     1081 gggtcgcttg cccgatccca ctaagggttc tgaccatttg agagatgtgt ttggcaaagc
     1141 tatggggctt actgaccaag atatcgttgc tctatctggg ggtcacacta ttggagctgc
     1201 acacaaggag cgttctggat ttgagggtcc ctggacctct aatcctctta ttttcgacaa
     1261 ctcatacttc acggagttgt tgagtggtga gaaggaaggt ctccttcagc taccttctga
     1321 caaggctctt ttgtctgacc ctgtattccg ccctctcgtt gacaaatatg cagcggacga
     1381 agatgccttc tttgctgatt acgctgaggc tcaccaaaag ctttccgagc ttgggtttgc
     1441 tgatgccctg gaagttctgt tccaggggcc ctcaaaatat tcctccaagc cattattgga
     1501 tgtggaatta aataaaatcg ctgaaggatt agaactcact gcaaaaattt acaatgaaaa
     1561 gagaggacgc gaatggtggt ttaaaggttc aagaaatgaa gctcgtaaga cccaatgtga
     1621 agaattgcaa agagtatcca aagaaatcaa tactcttctg caaagtgaat ctttaacgaa
     1681 aagccaggta cttgaaaagg ttttaaattc tatagaaaca ttagataaaa ttgacagaga
     1741 catttctgcc gaatccaatt ggtttcaaag tactctgcaa aaggaagtca ggttatttcg
     1801 agatcaattg aaagatattt gccaattgga caagtatgcc tttaaatcaa caaaacttga
     1861 tgaaatcatc tctctggaaa tggaagaaca atttcaaaag atacaagatc ctgctgttca
     1921 acaaattgtc agggacttgc cttctcattg ccacaatgat gaagcaattt aatgagtcga
     1981 cattaattaa cgcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt
     2041 gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc
     2101 ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag
     2161 gaatgcaagg tctgttgaat gtcgtgaagg aagtagttcc tctggaagct tcttgaagac
     2221 aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc
     2281 tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc
     2341 acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca
     2401 aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt
     2461 gcacatgctt tacatgtatt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg
     2521 gggacgtggt tttcctttga aaaacacgat gataatatgg ccacaaccat ggtgagcaag
     2581 ggcgaggagg ataacatggc ctctctccca gcgacacatg agttacacat ctttggctcc
     2641 atcaacggtg tggactttga catggtgggt cagggcaccg gcaatccaaa tgatggttat
     2701 gaggagttaa acctgaagtc caccaagggt gacctccagt tctccccctg gattctggtc
     2761 cctcatatcg ggtatggctt ccatcagtac ctgccctacc ctgacgggat gtcgcctttc
     2821 caggccgcca tggtagatgg ctccggatac caagtccatc gcacaatgca gtttgaagat
     2881 ggtgcctccc ttactgttaa ctaccgctac acctacgagg gaagccacat caaaggagag
     2941 gcccaggtga aggggactgg tttccctgct gacggtcctg tgatgaccaa ctcgctgacc
     3001 gctgcggact ggtgcaggtc gaagaagact taccccaacg acaaaaccat catcagtacc
     3061 tttaagtgga gttacaccac tggaaatggc aagcgctacc ggagcactgc gcggaccacc
     3121 tacacctttg ccaagccaat ggcggctaac tatctgaaga accagccgat gtacgtgttc
     3181 cgtaagacgg agctcaagca ctccaagacc gagctcaact tcaaggagtg gcaaaaggcc
     3241 tttaccgatg tgatgggcat ggacgagctg tacaagtagg acccagcttt cttgtacaaa
     3301 gttggcatta taagaaagca ttgcttatca atttgttgca acgaacaggt cactatcagt
     3361 caaaataaaa tcattatttg ccatccagct gatatcccct atagtgagtc gtattacatg
     3421 gtcatagctg tttcctggca gctctggccc gtgtctcaaa atctctgatg ttacattgca
     3481 caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa cagtaataca
     3541 aggggtgtta tgagccatat tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg
     3601 gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc aggtgcgaca
     3661 atctatcgct tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt
     3721 agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac ggaatttatg
     3781 cctcttccga ccatcaccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3841 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3901 gggtctgacg ctcagtggaa cgacgcgtaa ctcacgttaa gggattttgg tcatgagctt
     3961 gcgccgtccc gtcaagtcag cgtaatgctc tgccagtgtt acaaccaatt aaccaattct
     4021 gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     4081 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     4141 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     4201 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     4261 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     4321 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     4381 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     4441 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     4501 tattcttcta atacctggaa tgctgttttt ccggggatcg cagtggtgag taaccatgca
     4561 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     4621 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     4681 aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg
     4741 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     4801 ggcctggagc aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt
     4861 atgtaagcag acagttttat tgttcatgat gatatatttt tatcttgtgc aatgtaacat
     4921 cagagatttt gagacacaac gtggctttgt tgaataaatc gaacttttgc tgagttgaag
     4981 gatcagtcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5041 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5101 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5161 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     5221 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5281 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5341 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5401 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5461 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5521 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5581 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5641 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5701 ttgctcacat gtt
//
