LOCUS       sequence-338493-        7230 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses membrane targeted (palmitoylation) APEX2 and mRuby2 as 
            separate proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7230)
  TITLE     Expression plasmids combining APEX2 with various fluorophores and 
            targeting sequences
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7230)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAAV_Ef1a_FLEX_mAPEX2_P2A_mRuby2
FEATURES             Location/Qualifiers
     source          1..7230
                     /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    1437..1470
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1515..1548
                     /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             complement(1560..2270)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     CDS             complement(2331..2387)
                     /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(2397..3146)
                     /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="MGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAG
                     TFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVA
                     VEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGH
                     TIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVD
                     KYAADEDAFFADYAEAHQKLSELGFADA"
     protein_bind    complement(3215..3248)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(3299..3332)
                     /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)."
     misc_feature    3357..3945
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3410..3430)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3828..3839)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3977..4453
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4176..4195)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4493..4633
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4493..4622
                     /label=AAV2 ITR
     rep_origin      4708..5163
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4795..4814)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5005..5026
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5180..5199)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5299..5321
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5359..5377)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5445..5549
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5550..5618,5619..6410)
                     /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(5768..5787)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6581..7169
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7070..7089
                     /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 gtaccgccac catgtctaga gtcgacataa
     1441 cttcgtataa tgtatgctat acgaagttat gcagaatggt agcttaggct gctattagca
     1501 atatgaaacc tcttataact tcgtataaag tatcctatac gaagttatgt taattaatac
     1561 ttgtacagct cgtccatccc accaccaagc ccggcgaact tggcaactgc gtgttcgcgt
     1621 tgtactacga acatttcatt gtcactttcc tctaaccttt ccaggcggtg atcaacggca
     1681 tggataccgg gcatcttgat gttcccgacg gtcttttttg acctgtaagt tgttacgaaa
     1741 gagcaagaca gatggccacc accatcaact ttcagtgcca tatgagtgta tcccctcaga
     1801 ccaccatctg ctggatacat catctctgta ttaggctccc aacccttggt cttcttctgc
     1861 atcacgggac cattggaggg aaagtttacc cctctgactt ggacgtggta aacgagacag
     1921 ccatcctcaa ggctggtgtc ctgcatgacg gtgacgactc caccatcttc gtatctcgta
     1981 actctttccc aagtaaaacc ctcaggaaag gactgtttaa agaaatcagg aatgcctttc
     2041 gggtacttga taaaagtacg gctgccatac atgaacgacg tggcaagaat gtcaaaggca
     2101 aatggcaggg gtcctccctc gatgactttg atcctcatgg tttgagttcc catgtacgga
     2161 ttgccttctc cttcacctgt gcatttgaat tggtggccgt tgaccgaacc ttccatgacc
     2221 accttcatac gcatattttc cttgatcagc tcttcgccct tagacaccat catgatcttt
     2281 tggtcctcat cattcttttc aacctgtttg gttcttctca tacagcacag gggtccaggg
     2341 ttctcctcca cgtctccagc ctgcttcagc aggctgaagt tagtagctcc ggatccggca
     2401 tcagcaaacc caagctcgga aagcttttgg tgagcctcag cgtaatcagc aaagaaggca
     2461 tcttcgtccg ctgcatattt gtcaacgaga gggcggaata cagggtcaga caaaagagcc
     2521 ttgtcagaag gtagctgaag gagaccttcc ttctcaccac tcaacaactc cgtgaagtat
     2581 gagttgtcga aaataagagg attagaggtc cagggaccct caaatccaga acgctccttg
     2641 tgtgcagctc caatagtgtg acccccagat agagcaacga tatcttggtc agtaagcccc
     2701 atagctttgc caaacacatc tctcaaatgg tcagaaccct tagtgggatc gggcaagcga
     2761 ccctctggtg gtggctcagg cttgtcctct cttccagggt ggaatggaac cttaggtcca
     2821 cccgtgacct caacggcaac aacgccagcc aactggtaga aatcggcgta gctcaaaata
     2881 gggaactccg ccttgagtgg ctccaaaagc ctaacagcga tgtcaagacc gttgttagcg
     2941 ctgtgagcca gttcggcagg gtgcttgatg gttccgaagg gtccaccggt cttcgtgccc
     3001 ttgtcaaagg ttccagcaga gtggaatgcc aaacggagca ttagaggagc gcatctcttc
     3061 tcagcgatga agcctctgag cttcttcttc gccttctcaa cggcgtcctg gtaatcagca
     3121 ctcacagttg ggtaagactt tcccatgatc ttttggtcct catcattctt ttcaacctgt
     3181 ttggttcttc tcatacagca cagtatgcat gcccataact tcgtatagca tacattatac
     3241 gaagttattt gccttaaccc agaaattatc actgttattc tttagaatgg tgcaaagaat
     3301 aacttcgtat aggatacttt atacgaagtt atgaattcga tatcaagctt atcgataatc
     3361 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     3421 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     3481 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     3541 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     3601 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     3661 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     3721 gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct
     3781 atgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     3841 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     3901 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc
     3961 tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct
     4021 ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt
     4081 gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg
     4141 gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc
     4201 agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc
     4261 tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt
     4321 tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca
     4381 ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc
     4441 cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc
     4501 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     4561 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     4621 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4681 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     4741 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     4801 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4861 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4921 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     4981 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     5041 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     5101 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     5161 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     5221 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5281 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     5341 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     5401 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5461 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5521 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5581 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5641 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5701 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     5761 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     5821 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     5881 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     5941 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     6001 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     6061 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     6121 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6181 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6241 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     6301 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6361 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6421 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6481 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6541 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6601 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6661 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6721 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     6781 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     6841 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     6901 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     6961 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     7021 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     7081 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     7141 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     7201 tggccttttg ctggcctttt gctcacatgt
//
