LOCUS       sequence-426229-        7174 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses avitag-apex2-v5-fkbp8/biochemical assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7174)
  AUTHORS   Kwak C, Shin S, Park JS, Jung M, Nhung TTM, Kang MG, Lee C, Kwon TH,
            Park SK, Mun JY, Kim JS, Rhee HW
  TITLE     Contact-ID, a tool for profiling organelle contact sites, reveals 
            regulatory proteins of mitochondrial-associated membrane formation.
  JOURNAL   Proc Natl Acad Sci U S A. 2020 May 15. pii: 1916584117. doi: 
            10.1073/pnas.1916584117.
  PUBMED    32414919
REFERENCE   2  (bases 1 to 7174)
  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: "Proc Natl 
            Acad Sci U S A. 2020 May 15. pii: 1916584117. doi: 
            10.1073/pnas.1916584117."
COMMENT     Sequence Label: pCDNA5/avitag-apex2-v5-fkbp8
FEATURES             Location/Qualifiers
     source          1..7174
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             750..794
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             801..1547
                     /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             1554..1595
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2892..2909)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2898..3122
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3255..3274)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3406..3453
                     /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             3461..4483
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3461..4483
                     /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    4613..4734
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4650..4669)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4704..4723
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4783..4799)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4783..4799)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4796..4818)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4807..4823
                     /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(4831..4837,4838..4855,4856..4861))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4876..4897
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5014..5031)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5185..5773)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5265..5284)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5944..6735,6736..6804))
                     /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     6567..6586
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6805..6909)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6984..7003)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagatgg gcctgaacga catcttcgag gcccagaaga
      781 tcgagtggca cgaggctagc ggaaagtctt acccaactgt gagtgctgat taccaggacg
      841 ccgttgagaa ggcgaagaag aagctcagag gcttcatcgc tgagaagaga tgcgctcctc
      901 taatgctccg tttggcattc cactctgctg gaacctttga caagggcacg aagaccggtg
      961 gacccttcgg aaccatcaag caccctgccg aactggctca cagcgctaac aacggtcttg
     1021 acatcgctgt taggcttttg gagccactca aggcggagtt ccctattttg agctacgccg
     1081 atttctacca gttggctggc gttgttgccg ttgaggtcac gggtggacct aaggttccat
     1141 tccaccctgg aagagaggac aagcctgagc caccaccaga gggtcgcttg cccgatccca
     1201 ctaagggttc tgaccatttg agagatgtgt ttggcaaagc tatggggctt actgaccaag
     1261 atatcgttgc tctatctggg ggtcacacta ttggagctgc acacaaggag cgttctggat
     1321 ttgagggtcc ctggacctct aatcctctta ttttcgacaa ctcatacttc acggagttgt
     1381 tgagtggtga gaaggaaggt ctccttcagc taccttctga caaggctctt ttgtctgacc
     1441 ctgtattccg ccctctcgtt gataaatatg cagcggacga agatgccttc tttgctgatt
     1501 acgctgaggc tcaccaaaag ctttccgagc ttgggtttgc tgatgccggt accggcaagc
     1561 ccatccccaa ccccctgctg ggcctggaca gcaccgcggc cgcaatggca tcgtgtgctg
     1621 aaccctctga gccctctgcc ccactgcccg ccggggtccc accgctcgag gacttcgagg
     1681 tactggatgg ggttgaggat gcagagggtg aggaggaaga ggaggaggaa gaggaggaag
     1741 aggatgacct gagtgagctg ccaccgctgg aggacatggg acaacccccg gcggaggagg
     1801 ctgagcagcc tggggccctg gcccgagagt tccttgctgc catggagccc gagcccgccc
     1861 cagccccggc cccagaagag tggctggaca ttctggggaa cgggctgttg aggaagaaga
     1921 cgctggtccc agggccgcca ggttcgagcc gcccggtcaa gggccaggtg gtcaccgtac
     1981 atctgcagac gtcgctggag aatggcacac gggtgcagga ggagccggag ctggtgttca
     2041 ctctgggtga ctgtgacgtc atccaggccc tggatctcag tgtcccactc atggacgtgg
     2101 gggagacggc catggtcact gctgactcca agtactgcta cggcccccaa ggcagcagga
     2161 gcccatacat ccccccgcac gcggccctgt gcctggaggt gaccctgaag acggctgtgg
     2221 acgggcctga cctggagatg ctcacggggc aggagcgcgt ggccctggcc aaccggaagc
     2281 gggagtgcgg caacgcccac taccagcggg cggacttcgt cctggccgcc aactcctacg
     2341 acctcgccat caaggctatc acctccagcg ccaaagtgga catgacgttc gaggaggagg
     2401 cacagctcct gcagttgaag gtgaagtgtc tgaacaacct ggcggcctcg cagctgaagc
     2461 tcgaccacta ccgcgcagcc ctgcgctcct gcagccttgt gctggagcac cagccagaca
     2521 acatcaaggc tctcttccgc aagggcaagg tgctggccca gcagggggag tacagtgagg
     2581 ccatccccat cctgagggca gccctgaagc tggaaccttc caacaagacg atccacgcag
     2641 agctctcaaa gctggtgaag aagcatgcgg cgcagcggag cacggagacc gccttgtacc
     2701 ggaaaatgct gggcaacccc agccggctgc ctgctaagtg ccctggcaag ggtgcctggt
     2761 ccatcccatg gaagtggctg tttggggcga ctgctgttgc cttggggggt gtggcactct
     2821 ctgtggtcat cgctgccagg aactagggcg cgccctcgag tctagagggc ccgtttaaac
     2881 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2941 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     3001 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     3061 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     3121 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     3181 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     3241 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     3301 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     3361 cctcgacccc aaaaaacttg attagggtga tggttcacgt acctagaagt tcctattccg
     3421 aagttcctat tctctagaaa gtataggaac ttccttggcc aaaaagcctg aactcaccgc
     3481 gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct
     3541 ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct
     3601 gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc
     3661 atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac
     3721 ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact
     3781 gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag
     3841 ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg
     3901 tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga
     3961 caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg
     4021 ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa
     4081 tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga
     4141 ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta
     4201 cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg
     4261 cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg
     4321 ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca
     4381 aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag
     4441 tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagcacgtac tacgagattt
     4501 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     4561 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     4621 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4681 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4741 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     4801 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     4861 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     4921 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4981 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     5041 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     5101 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     5161 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5221 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5281 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5341 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5401 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5461 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5521 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5581 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5641 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5701 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5761 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5821 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5881 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5941 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6001 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6061 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     6121 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6181 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6241 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6301 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6361 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6421 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6481 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6541 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6601 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6661 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6721 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6781 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6841 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6901 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc
     6961 gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     7021 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
     7081 acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg
     7141 ctgcttcgcg atgtacgggc cagatatacg cgtt
//
