LOCUS       sequence-168982-        6609 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expressed FKBP25/FKBP3-V5-APEX2 in mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6609)
  AUTHORS   Lee SY, Lee H, Lee HK, Lee SW, Ha SC, Kwon T, Seo JK, Lee C, Rhee HW
  TITLE     Proximity-Directed Labeling Reveals a New Rapamycin-Induced 
            Heterodimer of FKBP25 and FRB in Live Cells.
  JOURNAL   ACS Cent Sci. 2016 Aug 24;2(8):506-16. doi: 
            10.1021/acscentsci.6b00137. Epub 2016 Aug 12.
  PUBMED    27610411
REFERENCE   2  (bases 1 to 6609)
  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: 
            "10.1021/acscentsci.6b00137"; journalName: "ACS Cent Sci"; date: 
            "2016-08-24- 24"; volume: "2"; issue: "8"; pages: "506-16"
COMMENT     Sequence Label: pCDNA5_FKBP25-V5-APEX2
FEATURES             Location/Qualifiers
     source          1..6609
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      988..997
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1677..1718
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1719..2465
                     /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             2466..2510
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     primer_bind     complement(2561..2578)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2567..2791
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2924..2943)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3075..3122
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3130..4152
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3130..4152
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4282..4403
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4319..4338)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4373..4392
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4452..4468)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4452..4468)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4465..4487)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4476..4492
                     /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(4500..4506,4507..4524,4525..4530))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4545..4566
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4683..4700)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4854..5442)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4934..4953)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5613..6404,6405..6473))
                     /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     6236..6255
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6474..6578)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtacca tggcggcggc cgttccacag cgggcgtgga
     1021 ccgtggagca gctgcgcagt gagcagctgc ccaagaagga cattatcaag tttctgcagg
     1081 aacacggttc agattcgttt cttgcagaac ataaattatt aggaaacatt aaaaatgtgg
     1141 ccaagacagc taacaaggac cacttggtta cagcctataa ccatcttttt gaaactaagc
     1201 gttttaaggg tactgaaagt ataagtaaag tgtctgagca agtaaaaaat gtgaagctta
     1261 atgaagataa acccaaagaa accaagtctg aagagaccct ggatgagggt ccaccaaaat
     1321 atactaaatc tgttctgaaa aagggagata aaaccaactt tcccaaaaag ggagatgttg
     1381 ttcactgctg gtatacagga acactacaag atgggactgt ttttgatact aatattcaaa
     1441 caagtgcaaa gaagaagaaa aatgccaagc ctttaagttt taaggtcgga gtaggcaaag
     1501 ttatcagagg atgggatgaa gctctcttga ctatgagtaa aggagaaaag gctcgactgg
     1561 agattgaacc agaatgggct tacggaaaga aaggacagcc tgatgccaaa attccaccaa
     1621 atgcaaaact cacttttgaa gtggaattag tggatattga taaggatccg ctagcaggca
     1681 agcccatccc caaccccctg ctgggcctgg acagcaccgg aaagtcttac ccaactgtga
     1741 gtgctgatta ccaggacgcc gttgagaagg cgaagaagaa gctcagaggc ttcatcgctg
     1801 agaagagatg cgctcctcta atgctccgtt tggcattcca ctctgctgga acctttgaca
     1861 agggcacgaa gaccggtgga cccttcggaa ccatcaagca ccctgccgaa ctggctcaca
     1921 gcgctaacaa cggtcttgac atcgctgtta ggcttttgga gccactcaag gcggagttcc
     1981 ctattttgag ctacgccgat ttctaccagt tggctggcgt tgttgccgtt gaggtcacgg
     2041 gtggacctaa ggttccattc caccctggaa gagaggacaa gcctgagcca ccaccagagg
     2101 gtcgcttgcc cgatcccact aagggttctg accatttgag agatgtgttt ggcaaagcta
     2161 tggggcttac tgaccaagat atcgttgctc tatctggggg tcacactatt ggagctgcac
     2221 acaaggagcg ttctggattt gagggtccct ggacctctaa tcctcttatt ttcgacaact
     2281 catacttcac ggagttgttg agtggtgaga aggaaggtct ccttcagcta ccttctgaca
     2341 aggctctttt gtctgaccct gtattccgcc ctctcgttga taaatatgca gcggacgaag
     2401 atgccttctt tgctgattac gctgaggctc accaaaagct ttccgagctt gggtttgctg
     2461 atgccggcct gaacgacatc ttcgaggccc agaagatcga gtggcacgag taatgagcgg
     2521 ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
     2581 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     2641 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     2701 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     2761 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     2821 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2881 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2941 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     3001 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     3061 ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact
     3121 tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag
     3181 ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc
     3241 ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac
     3301 aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt
     3361 gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc
     3421 acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc
     3481 atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg
     3541 caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat
     3601 gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc
     3661 gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat
     3721 ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc
     3781 gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg
     3841 ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga
     3901 tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg
     3961 gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga
     4021 tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc
     4081 gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg
     4141 gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4201 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     4261 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     4321 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4381 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     4441 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4501 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4561 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4621 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4681 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4741 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4801 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4861 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4921 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4981 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5041 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5101 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5161 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5221 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5281 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5341 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5401 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5461 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5521 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5581 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5641 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5701 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5761 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5821 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5881 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5941 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6001 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6061 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6121 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6181 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6241 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6301 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6361 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6421 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6481 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6541 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6601 cctgacgtc
//
