LOCUS       sequence-371412-        5681 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Middle entry vector for Gateway cloning containing APEX2 tagged to 
            mitochondria and nuclei, and cytoplasmic mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5681)
  AUTHORS   Agarwala S, Kim KY, Phan S, Ju S, Kong YE, Castillon GA, Bushong EA,
            Ellisman MH, Tamplin OJ
  TITLE     Defining the ultrastructure of the hematopoietic stem cell niche by 
            correlative light and electron microscopy.
  JOURNAL   Elife. 2022 Aug 9;11. pii: 64835. doi: 10.7554/eLife.64835.
  PUBMED    35943143
REFERENCE   2  (bases 1 to 5681)
  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: "Elife."; 
            date: "2022-08-9"; volume: "11. pii: 64835. doi"; pages: " 
            10.7554/eLife.64835"
COMMENT     Sequence Label: pME-APEX2-mCherry
FEATURES             Location/Qualifiers
     source          1..5681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    432..531
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             622..663
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             664..1410
                     /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             1420..1476
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1480..1503
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2281..2655
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     CDS             2665..2721
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2722..3432
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2867..2885)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3135..3154
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3329..3352
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(3434..3533)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3591..3607)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3591..3607)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3604..3626)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3615..3631
                     /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(3639..3645,3646..3663,3664..3669))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3684..3705
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3822..3839)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3993..4581)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4073..4092)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4759..5568)
                     /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     5482..5501
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5560..5579
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagagaatt cgagctcggt acctcgcgaa
      421 tacatctaga tcaaataatg attttatttt gactgatagt gacctgttcg ttgcaacaca
      481 ttgatgagca atgctttttt ataatgccaa ctttgtacaa aaaagcaggc tggacccacc
      541 atgctggcca cccgcgtgtt cagcctggtg ggcaagcgcg ccatcagcac cagcgtgtgc
      601 gtgcgcgccc acaaggatcc aggcaagccc atccccaacc ccctgctggg cctggacagc
      661 accggaaagt cttacccaac tgtgagtgct gattaccagg acgccgttga gaaggcgaag
      721 aagaagctca gaggcttcat cgctgagaag agatgcgctc ctctaatgct ccgtttggca
      781 ttccactctg ctggaacctt tgacaagggc acgaagaccg gtggaccctt cggaaccatc
      841 aagcaccctg ccgaactggc tcacagcgct aacaacggtc ttgacatcgc tgttaggctt
      901 ttggagccac tcaaggcgga gttccctatt ttgagctacg ccgatttcta ccagttggct
      961 ggcgttgttg ccgttgaggt cacgggtgga cctaaggttc cattccaccc tggaagagag
     1021 gacaagcctg agccaccacc agagggtcgc ttgcctgatc ccactaaggg ttctgaccat
     1081 ttgagagatg tgtttggcaa agctatgggg cttactgacc aagatatcgt tgctctatct
     1141 gggggtcaca ctattggagc tgcacacaag gagcgttctg gatttgaggg tccctggacc
     1201 tctaatcctc ttattttcga caactcatac ttcacggagt tgttgagtgg tgagaaggaa
     1261 ggtctccttc agctaccttc tgacaaggct cttttgtctg accctgtatt ccgccctctc
     1321 gttgataaat atgcagcgga cgaagatgcc ttctttgctg attacgctga ggctcaccaa
     1381 aagctttccg agcttgggtt tgctgatgcc ggaagcggag ctactaactt cagcctgctg
     1441 aagcaggctg gagacgtgga ggagaaccct ggacctatgg actacaagga tgacgacgat
     1501 aaggggaaat catacccaac agtgtccgca gactaccagg atgccgtgga gaaagccaag
     1561 aagaagctga gagggtttat cgcagaaaag aggtgcgcac ctctgatgct gagactggct
     1621 ttccacagcg caggcacctt tgacaaggga accaaaacag gcggaccctt tggaacaatc
     1681 aagcaccctg ctgaactggc acatagtgcc aacaatgggc tggacatcgc cgtgcggctg
     1741 ctggaacctc tgaaagcaga gttcccaatt ctgtcctacg ccgattttta tcagctggca
     1801 ggagtggtcg ctgtggaggt cactgggggc cccaaggtgc ctttccaccc aggacgggag
     1861 gacaaaccag aaccacctcc agaggggcgc ctgccagatc cgacaaaggg ctccgaccat
     1921 ctgcgagatg tgtttgggaa agctatgggc ctgactgacc aggatatcgt cgcactgtct
     1981 ggagggcaca ccattggcgc cgctcataag gaaaggtcag gcttcgaggg accctggaca
     2041 agcaaccccc tgattttcga caattcttac tttactgaac tgctgagtgg agagaaggaa
     2101 gggctgctgc agctgcccag cgacaaagcc ctgctgtccg atcccgtgtt cagacctctg
     2161 gtcgataagt atgcagccga cgaggatgct tttttcgcag attacgcaga agcacatcag
     2221 aagctgtcag aactgggatt tgccgacgcc tccggactca gatctggagg ctccggaggc
     2281 ccagagccag cgaagtctgc tcccgccccg aaaaagggct ccaagaaggc ggtgactaag
     2341 gcgcagaaga aaggcggcaa gaagcgcaag cgcagccgca aggagagcta ttccatctat
     2401 gtgtacaagg ttctgaagca ggtccaccct gacaccggca tttcgtccaa ggccatgggc
     2461 atcatgaatt cgtttgtgaa cgacattttc gagcgcatcg caggtgaggc ttcccgcctg
     2521 gcgcattaca acaagcgctc gaccatcacc tccagggaga tccagacggc cgtgcgcctg
     2581 ctgctgcctg gggagttggc caagcacgcc gtgtccgagg gtactaaggc catcaccaag
     2641 tacaccagcg ctaagggaag cggagctact aacttcagcc tgctgaagca ggctggagac
     2701 gtggaggaga accctggacc tatggtgagc aagggcgagg aggacaacat ggccatcatc
     2761 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
     2821 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
     2881 accaagggcg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc
     2941 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc
     3001 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
     3061 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac
     3121 ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag
     3181 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag
     3241 gacggcggcc actacgacgc cgaggtcaag accacctaca aggccaagaa gcccgtgcag
     3301 ctgcccggcg cctacaacgt caacatcaag ctggacatca cctcccacaa cgaggactac
     3361 accatcgtgg aacagtacga gcgcgccgag ggccgccact ccaccggcgg catggacgag
     3421 ctgtacaagt aagacccagc tttcttgtac aaagttggca ttataagaaa gcattgctta
     3481 tcaatttgtt gcaacgaaca ggtcactatc agtcaaaata aaatcattat ttgatcggat
     3541 cccgggcccg tcgactgcag aggcctgcat gcaagcttgg tgtaatcatg gtcatagctg
     3601 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     3661 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     3721 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3781 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3841 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3901 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     3961 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4021 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4081 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4141 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4201 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4261 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4321 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4381 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4441 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4501 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4561 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4621 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4681 tagatccttt taaattaaaa atgaagtttt aaatcaagcc caatctgaat aatgttacaa
     4741 ccaattaacc aattctgatt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     4801 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     4861 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     4921 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     4981 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     5041 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     5101 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     5161 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     5221 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gtttttccgg ggatcgcagt
     5281 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     5341 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     5401 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaagc gatagattgt
     5461 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     5521 gttggaattt aatcgcggcc tcgacgtttc ccgttgaata tggctcataa caccccttgt
     5581 attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt tatcttgtgc
     5641 aatgtaacat cagagatttt gagacacggg ccagagctgc a
//
