LOCUS       sequence-209369-        6450 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses V5_H2B_APEX2 in mammallian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6450)
  AUTHORS   Benhalevy D, Anastasakis DG, Hafner M
  TITLE     Proximity-CLIP provides a snapshot of protein-occupied RNA elements 
            in subcellular compartments.
  JOURNAL   Nat Methods. 2018 Dec;15(12):1074-1082. doi: 
            10.1038/s41592-018-0220-y. Epub 2018 Nov 26.
  PUBMED    30478324
REFERENCE   2  (bases 1 to 6450)
  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: 
            "10.1038/s41592-018-0220-y"; journalName: "Nat Methods"; date: 
            "2018-12"; volume: "15"; issue: "12"; pages: "1074-1082"
COMMENT     Sequence Label: pEXP(V5_H2B_APEX2)
FEATURES             Location/Qualifiers
     source          1..6450
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(324..343)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    475..522
                     /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             530..1552
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             530..1552
                     /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    1682..1803
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1719..1738)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1773..1792
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1852..1868)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1852..1868)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1865..1887)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1876..1892
                     /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(1900..1906,1907..1924,1925..1930))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1945..1966
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2083..2100)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2254..2842)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2334..2353)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3013..3804,3805..3873))
                     /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     3636..3655
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3874..3978)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4053..4072)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4244..4623
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4624..4827
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4778..4798
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    4829..4847
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4850..4868
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4878..4902
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             5002..5043
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    5041..5065
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             5068..5442
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="PEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKVL
                     KQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPG
                     ELAKHAVSEGTKAITKYTSAK"
     CDS             5464..5484
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             5494..6240
                     /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(6248..6272)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(6411..6428)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    join(6417..6450,1..191)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
       61 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
      121 ggggcaggac agcaaggggg aggattgtga agacaatagc aggcatgctg gggatgcggt
      181 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
      241 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
      301 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
      361 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
      421 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta cctagaagtt
      481 cctattccga agttcctatt ctctagaaag tataggaact tccttggcca aaaagcctga
      541 actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct
      601 gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg
      661 atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg
      721 gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga
      781 gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga
      841 aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga tcgctgcggc
      901 cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac
      961 tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt
     1021 gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc
     1081 cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct
     1141 gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc
     1201 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca
     1261 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta
     1321 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga
     1381 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg
     1441 gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact
     1501 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat agcacgtact
     1561 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     1621 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     1681 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     1741 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     1801 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     1861 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     1921 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     1981 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     2041 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     2101 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2161 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2221 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2281 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     2341 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     2401 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     2461 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     2521 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     2581 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     2641 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     2701 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     2761 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     2821 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     2881 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     2941 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3001 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3061 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3121 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3181 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3241 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3301 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     3361 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     3421 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     3481 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     3541 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     3601 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     3661 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     3721 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     3781 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     3841 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     3901 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     3961 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     4021 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     4081 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
     4141 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
     4201 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
     4261 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     4321 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     4381 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     4441 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     4501 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     4561 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     4621 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
     4681 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
     4741 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
     4801 cggtgggagg tctatataag cagagctctc cctatcagtg atagagatct ccctatcagt
     4861 gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca
     4921 cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccggac tctagcgttt
     4981 aaacttaagc ttggtaccat gggtaagcct atccctaacc ctctcctcgg tctcgattca
     5041 acaagtttgt acaaaaaagc aggcttccca gagccagcga agtctgctcc cgccccgaaa
     5101 aagggctcca agaaggcggt gactaaggcg cagaagaaag gcggcaagaa gcgcaagcgc
     5161 agccgcaagg agagctattc catctatgtg tacaaggttc tgaagcaggt ccaccctgac
     5221 accggcattt cgtccaaggc catgggcatc atgaattcgt ttgtgaacga cattttcgag
     5281 cgcatcgcag gtgaggcttc ccgcctggcg cattacaaca agcgctcgac catcacctcc
     5341 agggagatcc agacggccgt gcgcctgctg ctgcctgggg agttggccaa gcacgccgtg
     5401 tccgagggta ctaaggccat caccaagtac accagcgcta aggtttctct cgagccaacc
     5461 actgaggatc tgtactttca gagcgataac gatggaaagt cttacccaac tgtgagtgct
     5521 gattaccagg acgccgttga gaaggcgaag aagaagctca gaggcttcat cgctgagaag
     5581 agatgcgctc ctctaatgct ccgtttggca ttccactctg ctggaacctt tgacaagggc
     5641 acgaagaccg gtggaccctt cggaaccatc aagcaccctg ccgaactggc tcacagcgct
     5701 aacaacggtc ttgacatcgc tgttaggctt ttggagccac tcaaggcgga gttccctatt
     5761 ttgagctacg ccgatttcta ccagttggct ggcgttgttg ccgttgaggt cacgggtgga
     5821 cctaaggttc cattccaccc tggaagagag gacaagcctg agccaccacc agagggtcgc
     5881 ttgcccgatc ccactaaggg ttctgaccat ttgagagatg tgtttggcaa agctatgggg
     5941 cttactgacc aagatatcgt tgctctatct gggggtcaca ctattggagc tgcacacaag
     6001 gagcgttctg gatttgaggg tccctggacc tctaatcctc ttattttcga caactcatac
     6061 ttcacggagt tgttgagtgg tgagaaggaa ggtctccttc agctaccttc tgacaaggct
     6121 cttttgtctg accctgtatt ccgccctctc gttgacaaat atgcagcgga cgaagatgcc
     6181 ttctttgctg attacgctga ggctcaccaa aagctttccg agcttgggtt tgctgatgcc
     6241 taatagaacc cagctttctt gtacaaagtg gttgatatcc agcacagtgg cggccgctca
     6301 agtctagagg gcccgcggtt cgaaggtaac cggtaccgga tccgcggccg catagataac
     6361 tgatccagtg tgcctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     6421 gccttctagt tgccagccat ctgttgtttg
//
