LOCUS       sequence-254981-        6419 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express heterochromatin-localized CBX1-APEX2 fusion in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6419)
  AUTHORS   Padron A, Iwasaki S, Ingolia NT
  TITLE     Proximity RNA Labeling by APEX-Seq Reveals the Organization of 
            Translation Initiation Complexes and Repressive RNA Granules.
  JOURNAL   Mol Cell. 2019 Aug 22;75(4):875-887.e5. doi: 
            10.1016/j.molcel.2019.07.030.
  PUBMED    31442426
REFERENCE   2  (bases 1 to 6419)
  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.molcel.2019.07"; journalName: "Mol Cell"; date: 
            "2019-08-22- 22"; volume: "75"; issue: "4"; pages: "875-887.e5"
COMMENT     Sequence Label: pcDNA5/FRT/TO CBX1-APEX2
FEATURES             Location/Qualifiers
     source          1..6419
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(39..56)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    45..269
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(402..421)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    553..600
                     /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             608..1630
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             608..1630
                     /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    1760..1881
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1797..1816)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1851..1870
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1930..1946)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1930..1946)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1943..1965)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1954..1970
                     /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(1978..1984,1985..2002,2003..2008))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2023..2044
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2161..2178)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2332..2920)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2412..2431)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3091..3882,3883..3951))
                     /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     3714..3733
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3952..4056)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4131..4150)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4322..4701
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4702..4905
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4856..4876
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    4907..4925
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4928..4946
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4956..4980
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             5073..5096
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5649..6395
                     /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             6396..6416
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 gctcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg
       61 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
      121 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
      181 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
      241 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
      301 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
      361 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
      421 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
      481 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
      541 ttcacgtacc tagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc
      601 cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt
      661 cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt
      721 cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa
      781 agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga
      841 cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac
      901 gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat
      961 ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca
     1021 aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt
     1081 gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga
     1141 tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt
     1201 cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga
     1261 ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt
     1321 ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc
     1381 gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt
     1441 tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc
     1501 cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga
     1561 tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc
     1621 aaaggaatag cacgtactac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     1681 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     1741 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     1801 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     1861 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     1921 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     1981 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     2041 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2101 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     2161 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     2221 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2281 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2341 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2401 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2461 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2521 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     2581 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     2641 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     2701 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     2761 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     2821 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     2881 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     2941 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3001 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3061 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3121 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     3181 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     3241 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     3301 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     3361 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     3421 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     3481 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     3541 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     3601 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     3661 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     3721 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     3781 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     3841 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     3901 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     3961 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     4021 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     4081 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     4141 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     4201 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
     4261 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
     4321 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
     4381 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     4441 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     4501 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     4561 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     4621 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     4681 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
     4741 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     4801 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     4861 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctccc tatcagtgat
     4921 agagatctcc ctatcagtga tagagatcgt cgacgagctc gtttagtgaa ccgtcagatc
     4981 gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc
     5041 ctccggactc tagcgtttaa acttacacca tggactacaa agacgatgac gacaagggga
     5101 aaaaacaaaa caagaagaaa gtggaggagg tgctagaaga ggaggaagag gaatatgtgg
     5161 tggaaaaagt tctcgaccgt cgagtggtaa agggcaaagt ggagtacctc ctaaagtgga
     5221 agggattctc agatgaggac aacacatggg agccagaaga gaacctggat tgccccgacc
     5281 tcattgctga gtttctgcag tcacagaaaa cagcacatga gacagataaa tcagagggag
     5341 gcaagcgcaa agctgattct gattctgaag ataagggaga ggagagcaaa ccaaagaaga
     5401 agaaagaaga gtcagaaaag ccacgaggct ttgctcgagg tttggagccg gagcggatta
     5461 ttggagctac agactccagt ggagagctca tgttcctgat gaaatggaaa aactctgatg
     5521 aggctgacct ggtccctgcc aaggaagcca atgtcaagtg cccacaggtt gtcatatcct
     5581 tctatgagga aaggctgacg tggcattcct acccctcgga ggatgatgac aaaaaagatg
     5641 acaagaacgg aaagtcttac ccaactgtga gtgctgatta ccaggacgcc gttgagaagg
     5701 cgaagaagaa gctcagaggc ttcatcgctg agaagagatg cgctcctcta atgctccgtt
     5761 tggcattcca ctctgctgga acctttgaca agggcacgaa gaccggtgga cccttcggaa
     5821 ccatcaagca ccctgccgaa ctggctcaca gcgctaacaa cggtcttgac atcgctgtta
     5881 ggcttttgga gccactcaag gcggagttcc ctattttgag ctacgccgat ttctaccagt
     5941 tggctggcgt tgttgccgtt gaggtcacgg gtggacctaa ggttccattc caccctggaa
     6001 gagaggacaa gcctgagcca ccaccagagg gtcgcttgcc cgatcccact aagggttctg
     6061 accatttgag agatgtgttt ggcaaagcta tggggcttac tgaccaagat atcgttgctc
     6121 tatctggggg tcacactatt ggagctgcac acaaggagcg ttctggattt gagggtccct
     6181 ggacctctaa tcctcttatt ttcgacaact catacttcac ggagttgttg agtggtgaga
     6241 aggaaggtct ccttcagcta ccttctgaca aggctctttt gtctgaccct gtattccgcc
     6301 ctctcgttga caaatatgca gcggacgaag atgccttctt tgctgattac gctgaggctc
     6361 accaaaagct ttccgagctt gggtttgctg atgcccccaa gaaaaaacgc aaggtgtaa
//
