LOCUS       sequence-307434-        6377 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Triple-mutant CBX1 chromodomain (residues 20-73) tagged with 
            reversibly switchable FP Dronpa. Mutations V3E/K6E/D40S correspond 
            to positions V22/K25/D59 in full-length CBX1..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6377)
  AUTHORS   Hard R, Li N, He W, Ross B, Mo GCH, Peng Q, Stein RSL, Komives E, 
            Wang Y, Zhang J, Wang W
  TITLE     Deciphering and engineering chromodomain-methyllysine peptide 
            recognition.
  JOURNAL   Sci Adv. 2018 Nov 7;4(11):eaau1447. doi: 10.1126/sciadv.aau1447. 
            eCollection 2018 Nov.
  PUBMED    30417094
REFERENCE   2  (bases 1 to 6377)
  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: "Sci Adv."; 
            date: "2018-11-7"; volume: "4(11):eaau1447. doi"; pages: " 
            10.1126/sciadv.aau1447. eCollection 2018 Nov"
COMMENT     Sequence Label: pcDNA3-CBX1mut-Dronpa
FEATURES             Location/Qualifiers
     source          1..6377
                     /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"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             943..975
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(979..987,988..1002)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     regulatory      1009..1018
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1195..1869
                     /codon_start=1
                     /product="monomeric photoswitchable Dronpa-Green1 
                     fluorescent protein"
                     /label=Dronpa-Green1
                     /translation="MSVIKPDMKIKLRMEGAVNGHPFAIEGVGLGKPFEGKQSMDLKVK
                     EGGPLPFAYDILTTVFCYGNRVFAKYPENIVDYFKQSFPEGYSWERSMNYEDGGICNAT
                     NDITLDGDCYIYEIRFDGVNFPANGPVMQKRTVKWEPSTEKLYVRDGVLKGDVNMALSL
                     EGGGHYRCDFKTTYKAKKVVQLPDYHFVDHHIEIKSHDKDYSNVNLHEHAEAHSELPRQ
                     AK"
     promoter        complement(1930..1948)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1931..1948)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1968..1985)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1974..2198
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2244..2672
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2331..2350)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2541..2562
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2681..2701)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2686..3015
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2866..3001
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2928..2947
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3082..3876
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3136..3155)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3746..3765
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4050..4171
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4087..4106)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4141..4160
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4220..4236)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4220..4236)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4233..4255)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4244..4260
                     /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(4268..4274,4275..4292,4293..4298))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4313..4334
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4451..4468)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4622..5210)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4702..4721)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5381..6172,6173..6241))
                     /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     6004..6023
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6242..6346)
                     /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 ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag catgactggt
      961 ggacagcaaa tgggtcggga tctgtacgac gatgacgata aggatccggc caccatggaa
     1021 tatgaggtgg aagaggttct cgaccgtcga gtggtaaagg gcaaagtgga gtacctccta
     1081 aagtggaagg gattctcaga tgaggacaac acatgggagc cagaagagaa cctgagttgt
     1141 cccgatctca ttgctgagtt tctgcagtca cagaaaacag gaggtagtgg taccatgagt
     1201 gtgattaaac cagacatgaa gatcaagctg cgtatggaag gcgctgtaaa tggacacccg
     1261 ttcgcgattg aaggagttgg ccttgggaag cctttcgagg gaaaacagag tatggacctt
     1321 aaagtcaaag aaggcggacc tctgcctttc gcctatgaca tcttgacaac tgtgttctgt
     1381 tacggcaaca gggtattcgc caaataccca gaaaatatag tagactattt caagcagtcg
     1441 tttcctgagg gctactcttg ggaacgaagc atgaattacg aagacggggg catttgtaac
     1501 gcgacaaacg acataaccct ggatggtgac tgttatatct atgaaattcg atttgatggt
     1561 gtgaactttc ctgccaatgg tccagttatg cagaagagga ctgtgaaatg ggagccatcc
     1621 actgagaaat tgtatgtgcg tgatggagtg ctgaagggtg atgttaacat ggctctgtcg
     1681 cttgaaggag gtggccatta ccgatgtgac ttcaaaacta cttataaagc taagaaggtt
     1741 gtccagttgc cagactatca ctttgtggac caccacattg agattaaaag ccacgacaaa
     1801 gattacagta atgttaatct gcatgagcat gccgaagcgc attctgagct gccgaggcag
     1861 gccaagtaag aattctgcag atatccatca cactggcggc cgctcgagca tgcatctaga
     1921 gggccctatt ctatagtgtc acctaaatgc tagagctcgc tgatcagcct cgactgtgcc
     1981 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2041 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2101 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2161 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2221 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2281 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2341 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2401 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2461 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2521 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2581 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2641 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     2701 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     2761 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2821 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     2881 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     2941 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     3001 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     3061 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     3121 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     3181 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     3241 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     3301 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     3361 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     3421 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     3481 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     3541 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     3601 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     3661 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     3721 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     3781 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     3841 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     3901 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     3961 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     4021 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     4081 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4141 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     4201 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4261 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4321 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4381 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4441 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4501 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4561 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4621 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4681 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4741 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4801 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4861 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4921 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4981 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5041 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5101 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5161 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5221 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5281 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5341 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5401 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5461 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5521 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5581 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5641 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5701 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5761 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5821 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5881 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5941 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6001 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6061 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6121 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6181 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6241 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6301 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6361 aagtgccacc tgacgtc
//
