LOCUS       sequence-225174-        6309 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Plasmid name in publication: pAAV-Matrix-dAPEX2. Peroxidase reporter
            for multiplexed electron microscopy labeling.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6309)
  AUTHORS   Zhang Q, Lee WA, Paul DL, Ginty DD
  TITLE     Multiplexed peroxidase-based electron microscopy labeling enables 
            simultaneous visualization of multiple cell types.
  JOURNAL   Nat Neurosci. 2019 Mar 18. pii: 10.1038/s41593-019-0358-7. doi: 
            10.1038/s41593-019-0358-7.
  PUBMED    30886406
REFERENCE   2  (bases 1 to 6309)
  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: "Nat 
            Neurosci. 2019 Mar 18. pii: 10.1038/s41593-019-0358-7. doi: 
            10.1038/s41593-019-0358-7."
COMMENT     Sequence Label: pAAV-COX4-dAPEX2
FEATURES             Location/Qualifiers
     source          1..6309
                     /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)."
     CDS             1567..1608
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1609..2355
                     /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="GKSYPTVSADYQKAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPEVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     protein_bind    2378..2411
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2436..3024
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2489..2509)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2907..2918)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3056..3532
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3255..3274)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3572..3712
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3572..3701
                     /label=AAV2 ITR
     rep_origin      3787..4242
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3874..3893)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4084..4105
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4259..4278)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4378..4400
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4438..4456)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4524..4628
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4629..4697,4698..5489)
                     /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(4847..4866)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5660..6248
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6149..6168
                     /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 tatgctagcg ccaccatgct ggccacccgc
     1501 gtgttcagcc tggtgggcaa gcgcgccatc agcaccagcg tgtgcgtgcg cgcccacaag
     1561 gatccaggca agcccatccc caaccccctg ctgggcctgg acagcaccgg aaagtcttac
     1621 ccaactgtga gtgctgatta ccagaaggcc gttgagaagg cgaagaagaa gctcagaggc
     1681 ttcatcgctg agaagagatg cgctcctcta atgctccgtt tggcattcca ctctgctgga
     1741 acctttgaca agggcacgaa gaccggtgga cccttcggaa ccatcaagca ccctgccgaa
     1801 ctggctcaca gcgctaacaa cggtcttgac atcgctgtta ggcttttgga gccactcaag
     1861 gcggagttcc ctattttgag ctacgccgat ttctaccagt tggctggcgt tgttgccgtt
     1921 gaggtcacgg gtggacctga agttccattc caccctggaa gagaggacaa gcctgagcca
     1981 ccaccagagg gtcgcttgcc cgatcccact aagggttctg accatttgag agatgtgttt
     2041 ggcaaagcta tggggcttac tgaccaagat atcgttgctc tatctggggg tcacactatt
     2101 ggagctgcac acaaggagcg ttctggattt gagggtccct ggacctctaa tcctcttatt
     2161 ttcgacaact catacttcac ggagttgttg agtggtgaga aggaaggtct ccttcagcta
     2221 ccttctgaca aggctctttt gtctgaccct gtattccgcc ctctcgttga taaatatgca
     2281 gcggacgaag atgccttctt tgctgattac gctgaggctc accaaaagct ttccgagctt
     2341 gggtttgctg atgcctagtg tacaagtaag gcgcgccata acttcgtata atgtatgcta
     2401 tacgaagtta tgaattcgat atcaagctta tcgataatca acctctggat tacaaaattt
     2461 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     2521 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     2581 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     2641 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     2701 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     2761 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     2821 tgtcggggaa atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc
     2881 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     2941 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     3001 tctccctttg ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tctacgggtg
     3061 gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc
     3121 caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta
     3181 taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg
     3241 tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac
     3301 tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg
     3361 attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt
     3421 caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc
     3481 tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt
     3541 gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact
     3601 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     3661 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     3721 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     3781 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     3841 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     3901 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     3961 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     4021 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4081 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     4141 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     4201 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     4261 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     4321 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4381 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     4441 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     4501 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     4561 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     4621 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     4681 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     4741 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     4801 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     4861 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     4921 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     4981 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5041 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5101 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     5161 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     5221 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     5281 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     5341 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     5401 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     5461 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     5521 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     5581 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5641 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5701 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5761 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     5821 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5881 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5941 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6001 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6061 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6121 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6181 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6241 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6301 ctcacatgt
//
