LOCUS       sequence-361817-        7202 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent expression of cytosolic APEX .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7202)
  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 7202)
  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-APEX.NES-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..7202
                     /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(2361..2387)
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     CDS             complement(2394..3140)
                     /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(3141..3164)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    3187..3220
                     /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    3271..3304
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3329..3917
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3382..3402)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3800..3811)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3949..4425
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4148..4167)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4465..4605
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4465..4594
                     /label=AAV2 ITR
     rep_origin      4680..5135
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4767..4786)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4977..4998
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5152..5171)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5271..5293
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5331..5349)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5417..5521
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5522..5590,5591..6382)
                     /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(5740..5759)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6553..7141
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7042..7061
                     /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 cggatccgtc cagggtcagg cgctccaggg gaggcagctg cagggcatca
     2401 gcaaacccaa gctcggaaag cttttggtga gcctcagcgt aatcagcaaa gaaggcatct
     2461 tcgtccgctg catatttgtc aacgagaggg cggaatacag ggtcagacaa aagagccttg
     2521 tcagaaggta gctgaaggag accttccttc tcaccactca acaactccgt gaagtatgag
     2581 ttgtcgaaaa taagaggatt agaggtccag ggaccctcaa atccagaacg ctccttgtgt
     2641 gcagctccaa tagtgtgacc cccagataga gcaacgatat cttggtcagt aagccccata
     2701 gctttgccaa acacatctct caaatggtca gaacccttag tgggatcggg caagcgaccc
     2761 tctggtggtg gctcaggctt gtcctctctt ccagggtgga atggaacctt aggtccaccc
     2821 gtgacctcaa cggcaacaac gccagccaac tggtagaaat cggcgtagct caaaataggg
     2881 aactccgcct tgagtggctc caaaagccta acagcgatgt caagaccgtt gttagcgctg
     2941 tgagccagtt cggcagggtg cttgatggtt ccgaagggtc caccggtctt cgtgcccttg
     3001 tcaaaggttc cagcagagtg gaatgccaaa cggagcatta gaggagcgca tctcttctca
     3061 gcgatgaagc ctctgagctt cttcttcgcc ttctcaacgg cgtcctggta atcagcactc
     3121 acagttgggt aagactttcc cttatcgtcg tcatccttgt agtccatcgg cggcgcggtg
     3181 gctagcataa cttcgtataa agtatcctat acgaagttat ttgccttaac ccagaaatta
     3241 tcactgttat tctttagaat ggtgcaaaga ataacttcgt ataatgtatg ctatacgaag
     3301 ttatgaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa tttgtgaaag
     3361 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     3421 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     3481 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     3541 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     3601 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     3661 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     3721 gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc acctggattc tgcgcgggac
     3781 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     3841 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     3901 ttgggccgcc tccccgcatc gataccgagc gctgctcgag agatctacgg gtggcatccc
     3961 tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt gcccaccagc
     4021 cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt ctataatatt
     4081 atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac ctgtagggcc
     4141 tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct cactgcaatc
     4201 tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt gggattccag
     4261 gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg tttcaccata
     4321 ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg gcctcccaaa
     4381 ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat tttgtaggta
     4441 accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     4501 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     4561 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     4621 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     4681 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4741 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4801 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4861 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     4921 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4981 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     5041 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     5101 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     5161 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     5221 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     5281 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     5341 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     5401 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     5461 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5521 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     5581 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     5641 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     5701 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     5761 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     5821 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     5881 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     5941 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6001 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6061 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6121 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6181 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     6241 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     6301 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     6361 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     6421 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6481 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6541 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6601 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6661 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     6721 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6781 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     6841 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     6901 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     6961 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     7021 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7081 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7141 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7201 gt
//
