LOCUS       sequence-307435-        6413 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Triple-mutant CBX1 chromodomain (residues 20-73) tagged with 
            photoactivatable mCherry (PAmCherry). 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 6413)
  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 6413)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-PA-mCherry
FEATURES             Location/Qualifiers
     source          1..6413
                     /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..1905
                     /codon_start=1
                     /product="photoactivatable monomeric derivative of DsRed 
                     fluorescent protein"
                     /label=PAmCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHVFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFTWDILSPQFMYGSNAYVKHPADIPDYFKLSFPEGFKWERVMKF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEALSERMYPEDGALK
                     GEVKPRVKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNRKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1340..1358)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1608..1627
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(1966..1984)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1967..1984)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2004..2021)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2010..2234
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2280..2708
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2367..2386)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2577..2598
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2717..2737)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2722..3051
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2902..3037
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2964..2983
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3118..3912
                     /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(3172..3191)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3782..3801
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4086..4207
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4123..4142)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4177..4196
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4256..4272)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4256..4272)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4269..4291)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4280..4296
                     /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(4304..4310,4311..4328,4329..4334))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4349..4370
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4487..4504)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4658..5246)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4738..4757)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5417..6208,6209..6277))
                     /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     6040..6059
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6278..6382)
                     /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 taccatggtg
     1201 agcaagggcg aggaggataa catggccatc attaaggagt tcatgcgctt caaggtgcac
     1261 atggaggggt ccgtgaacgg ccacgtgttc gagatcgagg gcgagggcga gggccgcccc
     1321 tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcacc
     1381 tgggacatcc tgtcccctca attcatgtac ggctccaatg cctacgtgaa gcaccccgcc
     1441 gacatccccg actactttaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg
     1501 aaattcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggtgag
     1561 ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag
     1621 aagaagacca tgggctggga ggccctctcc gagcggatgt accccgagga cggcgccctg
     1681 aagggcgagg tcaagccgag agtgaagctg aaggacggcg gccactacga cgctgaggtc
     1741 aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaaccgc
     1801 aagttggaca tcacctcaca caacgaggac tacaccatcg tggaacagta cgaacgtgcc
     1861 gagggccgcc actccaccgg cggcatggac gagctgtaca agtaagaatt ctgcagatat
     1921 ccatcacact ggcggccgct cgagcatgca tctagagggc cctattctat agtgtcacct
     1981 aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     2041 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2101 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2161 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2221 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     2281 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2341 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2401 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2461 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2521 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2581 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2641 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2701 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     2761 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     2821 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     2881 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     2941 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     3001 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     3061 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     3121 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     3181 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     3241 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     3301 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     3361 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     3421 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     3481 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     3541 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     3601 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     3661 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     3721 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     3781 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     3841 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     3901 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     3961 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     4021 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     4081 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4141 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4201 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     4261 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4321 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4381 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4441 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     4501 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4561 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4621 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4681 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4741 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4801 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4861 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4921 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4981 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5041 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5101 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5161 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5221 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5281 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5341 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5401 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5461 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5521 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     5581 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     5641 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     5701 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     5761 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     5821 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5881 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5941 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6001 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6061 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6121 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6181 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6241 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6301 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6361 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
