LOCUS       sequence-361819-        7289 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent expression of membrane localized APEX.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7289)
  AUTHORS   Dumrongprechachan V, Salisbury RB, Soto G, Kumar M, MacDonald ML, 
            Kozorovitskiy Y
  TITLE     Cell-type and subcellular compartment-specific APEX2 proximity 
            labeling reveals activity-dependent nuclear proteome dynamics in the
            striatum.
  JOURNAL   Nat Commun. 2021 Aug 11;12(1):4855. doi: 10.1038/s41467-021-25144-y.
  PUBMED    34381044
REFERENCE   2  (bases 1 to 7289)
  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: "Nat 
            Commun."; date: "2021-08-11"; volume: "12(1):4855. doi"; pages: " 
            10.1038/s41467-021-25144-y"
COMMENT     Sequence Label: pAAV-EF1a-DIO-LckAPEX-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..7289
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1440..1473)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1524..1557)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1575..2285,2286..2288,2289..2291))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1575..2285,2286..2288,2289..2291))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1617..1638)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1966..1985
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2225..2246
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2292..2348)
                     /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             complement(2358..3104)
                     /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             complement(3105..3128)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      3260..3269
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3274..3307
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3358..3391
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3416..4004
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3469..3489)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3887..3898)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4036..4512
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4235..4254)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4552..4692
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4552..4681
                     /label=AAV2 ITR
     rep_origin      4767..5222
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4854..4873)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5064..5085
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5239..5258)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5358..5380
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5418..5436)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5504..5608
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5609..5677,5678..6469)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5827..5846)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6640..7228
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7129..7148
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa
     1441 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
     1501 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
     1561 gcgccttaac cggtcttgta cagctcgtcc atgccgagag tgatcccggc ggcggtcacg
     1621 aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag ggcggactgg
     1681 gtgctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg ggtgttctgc
     1741 tggtagtggt cggcgagctg cacgctgccg tcctcgatgt tgtggcggat cttgaagttc
     1801 accttgatgc cgttcttctg cttgtcggcc atgatataga cgttgtggct gttgtagttg
     1861 tactccagct tgtgccccag gatgttgccg tcctccttga agtcgatgcc cttcagctcg
     1921 atgcggttca ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg
     1981 tcgtccttga agaagatggt gcgctcctgg acgtagcctt cgggcatggc ggacttgaag
     2041 aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc actgcacgcc gtaggtcagg
     2101 gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat gaacttcagg
     2161 gtcagcttgc cgtaggtggc atcgccctcg ccctcgccgg acacgctgaa cttgtggccg
     2221 tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc cggtgaacag ctcctcgccc
     2281 ttgctcacca taggaccggg gttttcttcc acgtctcctg cttgctttaa cagagagaag
     2341 ttcgtggctc cggatccggc atcagcaaac ccaagctcgg aaagcttttg gtgagcctca
     2401 gcgtaatcag caaagaaggc atcttcgtcc gctgcatatt tgtcaacgag agggcggaat
     2461 acagggtcag acaaaagagc cttgtcagaa ggtagctgaa ggagaccttc cttctcacca
     2521 ctcaacaact ccgtgaagta tgagttgtcg aaaataagag gattagaggt ccagggaccc
     2581 tcaaatccag aacgctcctt gtgtgcagct ccaatagtgt gacccccaga tagagcaacg
     2641 atatcttggt cagtaagccc catagctttg ccaaacacat ctctcaaatg gtcagaaccc
     2701 ttagtgggat cgggcaagcg accctctggt ggtggctcag gcttgtcctc tcttccaggg
     2761 tggaatggaa ccttaggtcc acccgtgacc tcaacggcaa caacgccagc caactggtag
     2821 aaatcggcgt agctcaaaat agggaactcc gccttgagtg gctccaaaag cctaacagcg
     2881 atgtcaagac cgttgttagc gctgtgagcc agttcggcag ggtgcttgat ggttccgaag
     2941 ggtccaccgg tcttcgtgcc cttgtcaaag gttccagcag agtggaatgc caaacggagc
     3001 attagaggag cgcatctctt ctcagcgatg aagcctctga gcttcttctt cgccttctca
     3061 acggcgtcct ggtaatcagc actcacagtt gggtaagact ttcccttatc gtcgtcatcc
     3121 ttgtagtcgg tggcgaccgg tggatcccgg gcccgcggta ccgtcgactg cagaattcgt
     3181 ccgccgggat aatggcagtt ctcacacacg tcaatgttct ccatccagtc atcttcaggg
     3241 tttgagctgc agccacagcc catggtggct agcataactt cgtataaagt atcctatacg
     3301 aagttatttg ccttaaccca gaaattatca ctgttattct ttagaatggt gcaaagaata
     3361 acttcgtata atgtatgcta tacgaagtta tgaattcgat atcaagctta tcgataatca
     3421 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     3481 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     3541 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     3601 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     3661 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     3721 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     3781 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgccta
     3841 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     3901 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     3961 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgagcgct
     4021 gctcgagaga tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg
     4081 gaagttgcca ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg
     4141 tctgactagg tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg
     4201 caagttggga agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca
     4261 gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct
     4321 cagcctcccg agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt
     4381 ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag
     4441 gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc
     4501 ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac cgagcggccg caggaacccc
     4561 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     4621 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     4681 gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4741 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     4801 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt
     4861 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     4921 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     4981 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     5041 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc
     5101 tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct attggttaaa
     5161 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     5221 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     5281 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5341 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5401 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     5461 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5521 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5581 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5641 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5701 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     5761 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     5821 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     5881 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     5941 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     6001 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     6061 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     6121 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     6181 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     6241 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     6301 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6361 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6421 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6481 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6541 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6601 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6661 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6721 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6781 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6841 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     6901 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6961 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     7021 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     7081 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     7141 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     7201 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     7261 ggccttttgc tggccttttg ctcacatgt
//
