LOCUS       sequence-417379-        7875 bp    DNA     circular SYN 15-APR-2025
DEFINITION  SpyTag counterpart binding domain, SpyCatcher, fused to polycomb 
            repressive complex subunit EZH2, with GFP selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7875)
  AUTHORS   Swain T, Pflueger C, Freytag S, Poppe D, Pflueger J, Nguyen TV, Li 
            JK, Lister R
  TITLE     A modular dCas9-based recruitment platform for combinatorial 
            epigenome editing.
  JOURNAL   Nucleic Acids Res. 2023 Nov 24:gkad1108. doi: 10.1093/nar/gkad1108.
  PUBMED    38000387
REFERENCE   2  (bases 1 to 7875)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2023 Nov 24:gkad1108. doi: 10.1093/nar/gkad1108."
COMMENT     Sequence Label: LLP786_SpyCatcher-EZH2-FL-CO
FEATURES             Location/Qualifiers
     source          1..7875
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    6..230
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(join(240..950,951..953,954..956))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(240..950,951..953,954..956))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(282..303)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     631..650
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     890..911
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      953..962
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(981..1191)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      987..1122
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1041..1060)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(1248..1267)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1485..2663
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1716..2654
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2611..2631
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      2670..2679
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2682..2702
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2703..2774
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     polyA_signal    5501..5622
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5538..5557)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(5724..5740)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5724..5740)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5737..5759)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5748..5764
                     /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(5772..5778,5779..5796,5797..5802))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5817..5838
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5955..5972)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6126..6714)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6206..6225)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6885..7676,7677..7745))
                     /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     7508..7527
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7746..7850)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtccata gagcccaccg catccccagc atgcctgcta ttgtcttccc aatcctcccc
       61 cttgctgtcc tgccccaccc caccccccag aatagaatga cacctactca gacaatgcga
      121 tgcaatttcc tcattttatt aggaaaggac agtgggagtg gcaccttcca gggtcaagga
      181 aggcacgggg gaggggcaaa caacagatgg ctggcaacta gaaggcacag tcaactgccc
      241 ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc cagcaggacc
      301 atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg actgggtgct caggtagtgg
      361 ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta gtggtcggcg
      421 agctgcacgc tgccgtcctc gatgttgtgg cggatcttga agttcacctt gatgccgttc
      481 ttctgcttgt cggccatgat atagacgttg tggctgttgt agttgtactc cagcttgtgc
      541 cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg gttcaccagg
      601 gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag
      661 atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc gtgctgcttc
      721 atgtggtcgg ggtagcggct gaagcactgc acgccgtagg tcagggtggt cacgagggtg
      781 ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgtag
      841 gtggcatcgc cctcgccctc gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc
      901 agctcgacca ggatgggcac caccccggtg aacagctcct cgcccttgct caccatggtg
      961 gccgtacggg tggcgcgcgt agcctaggcc tccaaaaaag cctcctcact acttctggaa
     1021 tagctcagag gcagaggcgg cctcggcctc tgcataaata aaaaaaatta gtcagccatg
     1081 gggcggagaa tgggcggaac tgggcggagt taggggcggg atgggcggag ttaggggcgg
     1141 gactatggtt gctgactaat tgagatgcat gctttgcata cttctgcctg cctgaattca
     1201 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     1261 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     1321 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
     1381 ctgcttaggg ttaggcgttt tgcgtgcaaa gatggataaa gttttaaaca gagaggaatc
     1441 tttgcagcta atggaccttc taggtcttga aaggagtggg aattggctcc ggtgcccgtc
     1501 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt
     1561 gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc
     1621 tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg
     1681 ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg
     1741 ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc acctggctgc
     1801 agtacgtgat tcttgatccc gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt
     1861 gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg cgctggggcc
     1921 gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat aagtctctag
     1981 ccatttaaaa tttttgatga cctgctgcga cgcttttttt ctggcaagat agtcttgtaa
     2041 atgcgggcca agatctgcac actggtattt cggtttttgg ggccgcgggc ggcgacgggg
     2101 cccgtgcgtc ccagcgcaca tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa
     2161 tcggacgggg gtagtctcaa gctggccggc ctgctctggt gcctggcctc gcgccgccgt
     2221 gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg tgagcggaaa
     2281 gatggccgct tcccggccct gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag
     2341 agcgggcggg tgagtcaccc acacaaagga aaagggcctt tccgtcctca gccgtcgctt
     2401 catgtgactc cacggagtac cgggcgccgt ccaggcacct cgattagttc tcgagctttt
     2461 ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt ccccacactg
     2521 agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc ttggaatttg
     2581 ccctttttga gtttggatct tggttcattc tcaagcctca gacagtggtt caaagttttt
     2641 ttcttccatt tcaggtgtcg tgacgtacgg ccaccatggg tcctaagaaa aagaggaagg
     2701 tggattacaa ggatgacgac gataaggact ataaggacga tgatgacaag gactacaaag
     2761 atgatgacga taaagatagt gctacccata ttaaattctc aaaacgtgat gaggacggca
     2821 aagagttagc tggtgcaact atggagttgc gtgattcatc tggtaaaact attagtacat
     2881 ggatttcaga tggacaagtg aaagatttct acctgtatcc aggaaaatat acatttgtcg
     2941 aaaccgcagc accagacggt tatgaggtag caactgctat tacctttaca gttaatgagc
     3001 aaggtcaggt tactgtaaat ggcaaagcaa ctaaaggtga cgctcatatt agcgctggag
     3061 ggggtggctc cggcggagga ggctctggtg gcggagggag tggagcggcc gctatgggcc
     3121 agactgggaa gaaatctgag aagggaccag tttgttggcg gaagcgtgta aaatcagagt
     3181 acatgcgact gagacagctc aagaggttca gacgagctga tgaagtaaag agtatgttta
     3241 gttccaatcg tcagaaaatt ttggaaagaa cggaaatctt aaaccaagaa tggaaacagc
     3301 gaaggataca gcctgtgcac atcctgactt ctgtgagctc attgcgcggg actagggagt
     3361 gttcggtgac cagtgacttg gattttccaa cacaagtcat cccattaaag actctgaatg
     3421 cagttgcttc agtacccata atgtattctt ggtctcccct acagcagaat tttatggtgg
     3481 aagatgaaac tgttttacat aacattcctt atatgggaga tgaagtttta gatcaggatg
     3541 gtactttcat tgaagaacta ataaaaaatt atgatgggaa agtacacggg gatagagaat
     3601 gtgggtttat aaatgatgaa atttttgtgg agttggtgaa tgcccttggt caatataatg
     3661 atgatgacga tgatgatgat ggagacgatc ctgaagaaag agaagaaaag cagaaagatc
     3721 tggaggatca ccgagatgat aaagaaagcc gcccacctcg gaaatttcct tctgataaaa
     3781 tttttgaagc catttcctca atgtttccag ataagggcac agcagaagaa ctaaaggaaa
     3841 aatataaaga actcaccgaa cagcagctcc caggcgcact tcctcctgaa tgtaccccca
     3901 acatagatgg accaaatgct aaatctgttc agagagagca aagcttacac tcctttcata
     3961 cgcttttctg taggcgatgt tttaaatatg actgcttcct acatcgtaag tgcaattatt
     4021 cttttcatgc aacacccaac acttataagc ggaagaacac agaaacagct ctagacaaca
     4081 aaccttgtgg accacagtgt taccagcatt tggagggagc aaaggagttt gctgctgctc
     4141 tcaccgctga gcggataaag accccaccaa aacgtccagg aggccgcaga agaggacggc
     4201 ttcccaataa cagtagcagg cccagcaccc ccaccattaa tgtgctggaa tcaaaggata
     4261 cagacagtga tagggaagca gggactgaaa cggggggaga gaacaatgat aaagaagaag
     4321 aagagaagaa agatgaaact tcgagctcct ctgaagcaaa ttctcggtgt caaacaccaa
     4381 taaagatgaa gccaaatatt gaacctcctg agaatgtgga gtggagtggt gctgaagcct
     4441 caatgtttag agtcctcatt ggcacttact atgacaattt ctgtgccatt gctaggttaa
     4501 ttgggaccaa aacatgtaga caggtgtatg agtttagagt caaagaatct agcatcatag
     4561 ctccagctcc cgctgaggat gtggatactc ctccaaggaa aaagaagagg aaacaccggt
     4621 tgtgggctgc acactgcaga aagatacagc tgaaaaagga cggctcctct aaccatgttt
     4681 acaactatca accctgtgat catccacggc agccttgtga cagttcgtgc ccttgtgtga
     4741 tagcacaaaa tttttgtgaa aagttttgtc aatgtagttc agagtgtcaa aaccgctttc
     4801 cgggatgccg ctgcaaagca cagtgcaaca ccaagcagtg cccgtgctac ctggctgtcc
     4861 gagagtgtga ccctgacctc tgtcttactt gtggagccgc tgaccattgg gacagtaaaa
     4921 atgtgtcctg caagaactgc agtattcagc ggggctccaa aaagcatcta ttgctggcac
     4981 catctgacgt ggcaggctgg gggattttta tcaaagatcc tgtgcagaaa aatgaattca
     5041 tctcagaata ctgtggagag attattagcc aggacgaggc cgacaggagg ggcaaggtgt
     5101 acgacaagta catgtgctcc ttcctgttca acctgaacaa cgacttcgtg gtggacgcca
     5161 ccagaaaggg caacaagatc agattcgcca accacagcgt gaaccccaac tgctacgcca
     5221 aggtgatgat ggtgaacggc gaccacagaa tcggcatctt cgccaagagg gccatccaga
     5281 ccggcgagga gctgttcttc gactacaggt acagccaggc cgacgccctg aaatacgtgg
     5341 gcatcgagag ggagatggag atcccctgac gttgagtctc tgtttgtggt gactgatcat
     5401 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc
     5461 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta
     5521 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     5581 gcattctagt tgtggtttgt ccaaactcat caatgtatct taatgcgtcg agtgcattct
     5641 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     5701 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5761 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5821 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5881 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5941 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     6001 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     6061 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     6121 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     6181 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     6241 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     6301 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     6361 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     6421 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     6481 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     6541 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     6601 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6661 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6721 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6781 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6841 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6901 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6961 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     7021 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     7081 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     7141 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     7201 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     7261 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     7321 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     7381 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     7441 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     7501 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     7561 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7621 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7681 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7741 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7801 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7861 cgaaaagtgc cacct
//
