LOCUS       Exported                6609 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Plasmid name in publication: pAAV-DIO-IMS-dAPEX2. Peroxidase 
            reporter for multiplexed electron microscopy labeling.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-DIO-LACTB-dAPEX2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6609)
  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 6609)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6609
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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    1440..1473
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    1524..1557
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     CDS             complement(1580..2326)
                     /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"
     CDS             complement(2327..2368)
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    complement(2594..2627)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     protein_bind    complement(2678..2711)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    2736..3324
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2789..2809)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3207..3218)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3356..3832
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3555..3574)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3872..4012
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3872..4001
                     /label=AAV2 ITR
     rep_origin      4087..4542
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4174..4193)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4384..4405
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4559..4578)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4678..4700
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4738..4756)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4824..4928
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4929..5789
                     /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(5147..5166)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5960..6548
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6449..6468
                     /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 gcgccttact tgtacactag gcatcagcaa acccaagctc ggaaagcttt tggtgagcct
     1621 cagcgtaatc agcaaagaag gcatcttcgt ccgctgcata tttatcaacg agagggcgga
     1681 atacagggtc agacaaaaga gccttgtcag aaggtagctg aaggagacct tccttctcac
     1741 cactcaacaa ctccgtgaag tatgagttgt cgaaaataag aggattagag gtccagggac
     1801 cctcaaatcc agaacgctcc ttgtgtgcag ctccaatagt gtgaccccca gatagagcaa
     1861 cgatatcttg gtcagtaagc cccatagctt tgccaaacac atctctcaaa tggtcagaac
     1921 ccttagtggg atcgggcaag cgaccctctg gtggtggctc aggcttgtcc tctcttccag
     1981 ggtggaatgg aacttcaggt ccacccgtga cctcaacggc aacaacgcca gccaactggt
     2041 agaaatcggc gtagctcaaa atagggaact ccgccttgag tggctccaaa agcctaacag
     2101 cgatgtcaag accgttgtta gcgctgtgag ccagttcggc agggtgcttg atggttccga
     2161 agggtccacc ggtcttcgtg cccttgtcaa aggttccagc agagtggaat gccaaacgga
     2221 gcattagagg agcgcatctc ttctcagcga tgaagcctct gagcttcttc ttcgccttct
     2281 caacggcctt ctggtaatca gcactcacag ttgggtaaga ctttccggtg ctgtccaggc
     2341 ccagcagggg gttggggatg ggcttgcctg gatccttgga ctgaatgggg acggcgcccc
     2401 gcagcccgac caccagcttc gcgccgagag ccagccccag cccgagcccc aggccgccgg
     2461 cccaaccaag gcccagcaca ggcaggcccg gtcgcctgtg cgccccgcgc cgccctccgt
     2521 cccaggctgg gcctgcggtg gccgcagccc gagctgtcac gcttgacagg agccggtaca
     2581 tggtggcgct agcataactt cgtataaagt atcctatacg aagttatttg ccttaaccca
     2641 gaaattatca ctgttattct ttagaatggt gcaaagaata acttcgtata atgtatgcta
     2701 tacgaagtta tgaattcgat atcaagctta tcgataatca acctctggat tacaaaattt
     2761 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     2821 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     2881 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     2941 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     3001 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     3061 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     3121 tgtcggggaa atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc
     3181 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     3241 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     3301 tctccctttg ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tctacgggtg
     3361 gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc
     3421 caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta
     3481 taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg
     3541 tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac
     3601 tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg
     3661 attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt
     3721 caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc
     3781 tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt
     3841 gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact
     3901 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     3961 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     4021 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     4081 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4141 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     4201 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4261 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     4321 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4381 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     4441 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     4501 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     4561 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     4621 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     4681 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     4741 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     4801 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     4861 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     4921 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     4981 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     5041 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     5101 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     5161 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     5221 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5281 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5341 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5401 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     5461 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     5521 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     5581 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     5641 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     5701 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     5761 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     5821 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     5881 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5941 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6001 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6061 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6121 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6181 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6241 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6301 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6361 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6421 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6481 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6541 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6601 ctcacatgt
//
