LOCUS       sequence-116994-        6204 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector driving AKT-APEX2 (Human AKT-PH domain, 
            [marks PI(3,4)P2 and PI(3,4,5)P3] fused to EM peroxidase marker). 
            Also contains bicistronially expressed cytoplasmic red reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6204)
  AUTHORS   Ariotti N, Hall TE, Rae J, Ferguson C, McMahon KA, Martel N, Webb 
            RE, Webb RI, Teasdale RD, Parton RG
  TITLE     Modular Detection of GFP-Labeled Proteins for Rapid Screening by 
            Electron Microscopy in Cells and Organisms.
  JOURNAL   Dev Cell. 2015 Nov 23;35(4):513-25. doi: 
            10.1016/j.devcel.2015.10.016. Epub 2015 Nov 12.
  PUBMED    26585296
REFERENCE   2  (bases 1 to 6204)
  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"; doi: 
            "10.1016/j.devcel.2015.10.016"; journalName: "Dev Cell"; date: 
            "2015-11-23- 23"; volume: "35"; issue: "4"; pages: "513-25"
COMMENT     Sequence Label: AKT-APEX2-P2A-mKate2
FEATURES             Location/Qualifiers
     source          1..6204
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    108..132
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(632..656)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             661..1407
                     /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             1417..1473
                     /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             1492..2187
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
                     VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
                     LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHR"
     protein_bind    complement(2193..2213)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    2253..2387
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2277..2296)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2331..2350
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2500..2520)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2500..2518)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2539..2555)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2539..2555)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2552..2574)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2563..2579
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2587..2593,2594..2611,2612..2617))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2632..2653
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2770..2787)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2941..3529)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3021..3040)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3700..4491,4492..4560))
                     /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     4323..4342
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4561..4665)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4691..5146)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4828..4849)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5040..5059
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5220..6204
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgaattca aggatcaaca agtttgtaca
      121 aaaaagcagg ctgccaccat gagcgacgtg gctattgtga aggagggttg gctgcacaaa
      181 cgaggggagt acatcaagac ctggcggcca cgctacttcc tcctcaagaa tgatggcacc
      241 ttcattggct acaaggagcg gccgcaggat gtggaccaac gtgaggctcc cctcaacaac
      301 ttctctgtgg cgcagtgcca gctgatgaag acggagcggc cccggcccaa caccttcatc
      361 atccgctgcc tgcagtggac cactgtcatc gaacgcacct tccatgtgga gactcctgag
      421 gagcgggagg agtggacaac cgccatccag actgtggctg acggcctcaa gaagcaggag
      481 gaggaggaga tggacttccg gtcgggctca cccagtgaca actcaggggc tgaagagatg
      541 gaggtgtccc tggccaagcc caagcaccgc gtgaccatga acgagtttga gtacctgaag
      601 ctgctgggca agggcacttt cggcaaggtg gacccagctt tcttgtacaa agtggtcgac
      661 ggaaagtctt acccaactgt gagtgctgat taccaggacg ccgttgagaa ggcgaagaag
      721 aagctcagag gcttcatcgc tgagaagaga tgcgctcctc taatgctccg tttggcattc
      781 cactctgctg gaacctttga caagggcacg aagaccggtg gacccttcgg aaccatcaag
      841 caccctgccg aactggctca cagcgctaac aacggtcttg acatcgctgt taggcttttg
      901 gagccactca aggcggagtt ccctattttg agctacgccg atttctacca gttggctggc
      961 gttgttgccg ttgaggtcac gggtggacct aaggttccat tccaccctgg aagagaggac
     1021 aagcctgagc caccaccaga gggtcgcttg cccgatccca ctaagggttc tgaccatttg
     1081 agagatgtgt ttggcaaagc tatggggctt actgaccaag atatcgttgc tctatctggg
     1141 ggtcacacta ttggagctgc acacaaggag cgttctggat ttgagggtcc ctggacctct
     1201 aatcctctta ttttcgacaa ctcatacttc acggagttgt tgagtggtga gaaggaaggt
     1261 ctccttcagc taccttctga caaggctctt ttgtctgacc ctgtattccg ccctctcgtt
     1321 gacaaatatg cagcggacga agatgccttc tttgctgatt acgctgaggc tcaccaaaag
     1381 ctttccgagc ttgggtttgc tgatgccgga tccggagcca cgaacttctc tctgttaaag
     1441 caagcaggag acgtggaaga aaaccccggt cctgctcctg gaagcggtac cgtgagcgag
     1501 ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgaa caaccaccac
     1561 ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc
     1621 aaggcggtcg agggcggccc tctccccttc gccttcgaca tcctggctac cagcttcatg
     1681 tacggcagca aaaccttcat caaccacacc cagggcatcc ccgacttctt taagcagtcc
     1741 ttccccgagg gcttcacatg ggagagagtc accacatacg aagacggggg cgtgctgacc
     1801 gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg
     1861 gtgaacttcc catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggcctcc
     1921 accgagaccc tgtaccccgc tgacggcggc ctggaaggca gagccgacat ggccctgaag
     1981 ctcgtgggcg ggggccacct gatctgcaac ttgaagacca catacagatc caagaaaccc
     2041 gctaagaacc tcaagatgcc cggcgtctac tatgtggaca gaagactgga aagaatcaag
     2101 gaggccgaca aagagaccta cgtcgagcag cacgaggtgg ctgtggccag atactgcgac
     2161 ctccctagca aactggggca cagatgacta gtcaacttta ttatacatag ttgcactcga
     2221 gcctctagaa ctatagtgag tcgtattacg tagatccaga catgataaga tacattgatg
     2281 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     2341 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     2401 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa ttcgcggccg
     2461 cggcgccaat gcattgggcc cggtacccag cttttgttcc ctttagtgag ggttaattgc
     2521 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     2581 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     2641 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     2701 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     2761 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     2821 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     2881 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     2941 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3001 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     3061 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     3121 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     3181 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     3241 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     3301 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     3361 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     3421 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     3481 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     3541 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     3601 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     3661 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     3721 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     3781 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     3841 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     3901 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     3961 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     4021 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     4081 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     4141 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     4201 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     4261 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     4321 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     4381 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     4441 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     4501 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     4561 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     4621 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     4681 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     4741 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     4801 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     4861 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     4921 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     4981 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     5041 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     5101 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
     5161 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagtcg
     5221 accatagcca attcaatatg gcgtatatgg actcatgcca attcaatatg gtggatctgg
     5281 acctgtgcca attcaatatg gcgtatatgg actcgtgcca attcaatatg gtggatctgg
     5341 accccagcca attcaatatg gcggacttgg caccatgcca attcaatatg gcggacttgg
     5401 cactgtgcca actggggagg ggtctacttg gcacggtgcc aagtttgagg aggggtcttg
     5461 gccctgtgcc aagtccgcca tattgaattg gcatggtgcc aataatggcg gccatattgg
     5521 ctatatgcca ggatcaatat ataggcaata tccaatatgg ccctatgcca atatggctat
     5581 tggccaggtt caatactatg tattggccct atgccatata gtattccata tatgggtttt
     5641 cctattgacg tagatagccc ctcccaatgg gcggtcccat ataccatata tggggcttcc
     5701 taataccgcc catagccact cccccattga cgtcaatggt ctctatatat ggtctttcct
     5761 attgacgtca tatgggcggt cctattgacg tatatggcgc ctcccccatt gacgtcaatt
     5821 acggtaaatg gcccgcctgg ctcaatgccc attgacgtca ataggaccac ccaccattga
     5881 cgtcaatggg atggctcatt gcccattcat atccgttctc acgcccccta ttgacgtcaa
     5941 tgacggtaaa tggcccactt ggcagtacat caatatctat taatagtaac ttggcaagta
     6001 cattactatt ggaaggacgc cagggtacat tggcagtact cccattgacg tcaatggcgg
     6061 taaatggccc gcgatggctg ccaagtacat ccccattgac gtcaatgggg aggggcaatg
     6121 acgcaaatgg gcgttccatt gacgtaaatg ggcggtaggc gtgcctaatg ggaggtctat
     6181 ataagcaatg ctcgtttagg gaac
//
