LOCUS       sequence-173703-        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"
     enhancer        118..497
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        498..701
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     652..672
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    703..721
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    724..742
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     752..776
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      871..880
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1560..1601
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1602..2348
                     /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             2349..2393
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     primer_bind     complement(2444..2461)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2450..2674
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2807..2826)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2958..3005
                     /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             3013..4035
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3013..4035
                     /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    4165..4286
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4202..4221)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4256..4275
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4335..4351)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4335..4351)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4348..4370)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4359..4375
                     /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(4383..4389,4390..4407,4408..4413))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4428..4449
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4566..4583)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4737..5325)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4817..4836)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5496..6287,6288..6356))
                     /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     6119..6138
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6357..6461)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6536..6555)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
       61 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      121 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      181 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      241 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      301 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      361 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      421 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      481 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      541 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      601 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      661 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctccctatc agtgatagag
      721 atctccctat cagtgataga gatcgtcgac gagctcgttt agtgaaccgt cagatcgcct
      781 ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctcc
      841 ggactctagc gtttaaactt aagcttggta ccatggcggc ggccgttcca cagcgggcgt
      901 ggaccgtgga gcagctgcgc agtgagcagc tgcccaagaa ggacattatc aagtttctgc
      961 aggaacacgg ttcagattcg tttcttgcag aacataaatt attaggaaac attaaaaatg
     1021 tggccaagac agctaacaag gaccacttgg ttacagccta taaccatctt tttgaaacta
     1081 agcgttttaa gggtactgaa agtataagta aagtgtctga gcaagtaaaa aatgtgaagc
     1141 ttaatgaaga taaacccaaa gaaaccaagt ctgaagagac cctggatgag ggtccaccaa
     1201 aatatactaa atctgttctg aaaaagggag ataaaaccaa ctttcccaaa aagggagatg
     1261 ttgttcactg ctggtataca ggaacactac aagatgggac tgtttttgat actaatattc
     1321 aaacaagtgc aaagaagaag aaaaatgcca agcctttaag ttttaaggtc ggagtaggca
     1381 aagttatcag aggatgggat gaagctctct tgactatgag taaaggagaa aaggctcgac
     1441 tggagattga accagaatgg gcttacggaa agaaaggaca gcctgatgcc aaaattccac
     1501 caaatgcaaa actcactttt gaagtggaat tagtggatat tgataaggat ccgctagcag
     1561 gcaagcccat ccccaacccc ctgctgggcc tggacagcac cggaaagtct tacccaactg
     1621 tgagtgctga ttaccaggac gccgttgaga aggcgaagaa gaagctcaga ggcttcatcg
     1681 ctgagaagag atgcgctcct ctaatgctcc gtttggcatt ccactctgct ggaacctttg
     1741 acaagggcac gaagaccggt ggacccttcg gaaccatcaa gcaccctgcc gaactggctc
     1801 acagcgctaa caacggtctt gacatcgctg ttaggctttt ggagccactc aaggcggagt
     1861 tccctatttt gagctacgcc gatttctacc agttggctgg cgttgttgcc gttgaggtca
     1921 cgggtggacc taaggttcca ttccaccctg gaagagagga caagcctgag ccaccaccag
     1981 agggtcgctt gcccgatccc actaagggtt ctgaccattt gagagatgtg tttggcaaag
     2041 ctatggggct tactgaccaa gatatcgttg ctctatctgg gggtcacact attggagctg
     2101 cacacaagga gcgttctgga tttgagggtc cctggacctc taatcctctt attttcgaca
     2161 actcatactt cacggagttg ttgagtggtg agaaggaagg tctccttcag ctaccttctg
     2221 acaaggctct tttgtctgac cctgtattcc gccctctcgt tgataaatat gcagcggacg
     2281 aagatgcctt ctttgctgat tacgctgagg ctcaccaaaa gctttccgag cttgggtttg
     2341 ctgatgccgg cctgaacgac atcttcgagg cccagaagat cgagtggcac gagtaatgag
     2401 cggccgctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct
     2461 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     2521 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2581 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2641 agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     2701 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2761 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2821 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2881 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2941 gatggttcac gtacctagaa gttcctattc cgaagttcct attctctaga aagtatagga
     3001 acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa
     3061 aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc
     3121 agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc
     3181 tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg
     3241 cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt
     3301 gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag
     3361 gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga
     3421 ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc
     3481 catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct
     3541 ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg
     3601 gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg
     3661 agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg
     3721 tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca
     3781 ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc
     3841 ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc
     3901 cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg
     3961 accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg
     4021 agggcaaagg aatagcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4081 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4141 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     4201 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4261 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     4321 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4381 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4441 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4501 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4561 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4621 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4681 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4741 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4801 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4861 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4921 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4981 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5041 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5101 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5161 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5221 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5281 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5341 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5401 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5461 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5521 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5581 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5641 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5701 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5761 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5821 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5881 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5941 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6001 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6061 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6121 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6181 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6241 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6301 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6361 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6421 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6481 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
     6541 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     6601 ctgagtagt
//
