LOCUS       sequence-338504-        7164 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a membrane targeted (farnesylation) APEX2-eGFP fusion 
            protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7164)
  TITLE     Expression plasmids combining APEX2 with various fluorophores and 
            targeting sequences
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7164)
  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            "Unpublished"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_Efla_FLEX_APEX2-eGFPf
FEATURES             Location/Qualifiers
     source          1..7164
                     /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(join(1638..2348,2349..2351))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(1638..2348,2349..2351))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1680..1701)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2029..2048
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2288..2309
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2391..3137)
                     /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"
     protein_bind    complement(3149..3182)
                     /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(3233..3266)
                     /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    3291..3879
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3344..3364)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3762..3773)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3911..4387
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4110..4129)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4427..4567
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4427..4556
                     /label=AAV2 ITR
     rep_origin      4642..5097
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4729..4748)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4939..4960
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5114..5133)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5233..5255
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5293..5311)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5379..5483
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5484..5552,5553..6344)
                     /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(5702..5721)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6515..7103
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7004..7023
                     /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 taattaatat
     1561 caggagagca cacacttgca gctcatgcag ccggggccac tctcatcagg agggttcagc
     1621 ttactcacgc gtgcactctt gtacagctcg tccatgccga gagtgatccc ggcggcggtc
     1681 acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac
     1741 tgggtgctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc
     1801 tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag
     1861 ttcaccttga tgccgttctt ctgcttgtcg gccatgatat agacgttgtg gctgttgtag
     1921 ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc
     1981 tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg
     2041 ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg
     2101 aagaagtcgt gctgcttcat gtggtcgggg tagcggctga agcactgcac gccgtaggtc
     2161 agggtggtca cgagggtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc
     2221 agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg
     2281 ccgtttacgt cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg
     2341 cccttgctca cgcccgagcc cgaggtcgag cccgagccct tggcgcgccc ggcatcagca
     2401 aacccaagct cggaaagctt ttggtgagcc tcagcgtaat cagcaaagaa ggcatcttcg
     2461 tccgctgcat atttgtcaac gagagggcgg aatacagggt cagacaaaag agccttgtca
     2521 gaaggtagct gaaggagacc ttccttctca ccactcaaca actccgtgaa gtatgagttg
     2581 tcgaaaataa gaggattaga ggtccaggga ccctcaaatc cagaacgctc cttgtgtgca
     2641 gctccaatag tgtgaccccc agatagagca acgatatctt ggtcagtaag ccccatagct
     2701 ttgccaaaca catctctcaa atggtcagaa cccttagtgg gatcgggcaa gcgaccctct
     2761 ggtggtggct caggcttgtc ctctcttcca gggtggaatg gaaccttagg tccacccgtg
     2821 acctcaacgg caacaacgcc agccaactgg tagaaatcgg cgtagctcaa aatagggaac
     2881 tccgccttga gtggctccaa aagcctaaca gcgatgtcaa gaccgttgtt agcgctgtga
     2941 gccagttcgg cagggtgctt gatggttccg aagggtccac cggtcttcgt gcccttgtca
     3001 aaggttccag cagagtggaa tgccaaacgg agcattagag gagcgcatct cttctcagcg
     3061 atgaagcctc tgagcttctt cttcgccttc tcaacggcgt cctggtaatc agcactcaca
     3121 gttgggtaag actttcctcc atgggcccat aacttcgtat agcatacatt atacgaagtt
     3181 atttgcctta acccagaaat tatcactgtt attctttaga atggtgcaaa gaataacttc
     3241 gtataggata ctttatacga agttatgaat tcgatatcaa gcttatcgat aatcaacctc
     3301 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     3361 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     3421 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     3481 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     3541 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     3601 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     3661 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctatgttg
     3721 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     3781 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     3841 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccga gcgctgctcg
     3901 agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt
     3961 tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga
     4021 ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt
     4081 tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc
     4141 acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc
     4201 tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg
     4261 gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat
     4321 ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc
     4381 tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga acccctagtg
     4441 atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag
     4501 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc
     4561 ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     4621 atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     4681 tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct cctttcgctt
     4741 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     4801 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg
     4861 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     4921 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aactctatct
     4981 cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg ttaaaaaatg
     5041 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat
     5101 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
     5161 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     5221 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     5281 cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     5341 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     5401 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     5461 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     5521 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     5581 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     5641 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     5701 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     5761 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     5821 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     5881 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     5941 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     6001 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     6061 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6121 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6181 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     6241 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     6301 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     6361 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     6421 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     6481 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     6541 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     6601 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     6661 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     6721 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     6781 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     6841 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     6901 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     6961 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     7021 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     7081 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     7141 tttgctggcc ttttgctcac atgt
//
