LOCUS       Exported                9323 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses MCP (MS2 Coat Protein) fusion to Ezh2 in mammalian cells, 
            lentiviral backbone.
ACCESSION   .
VERSION     .
KEYWORDS    NLS-HA-2xMCP-Ezh2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9323)
  AUTHORS   O'Geen H, Bates SL, Carter SS, Nisson KA, Halmai J, Fink KD, Rhie 
            SK, Farnham PJ, Segal DJ
  TITLE     Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in
            a context-dependent manner.
  JOURNAL   Epigenetics Chromatin. 2019 May 3;12(1):26. doi: 
            10.1186/s13072-019-0275-8.
  PUBMED    31053162
REFERENCE   2  (bases 1 to 9323)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9323
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2218..2616
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     CDS             2631..2651
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2655..2681
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2682..2693
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_feature    5713..6301
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5766..5786)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6184..6195)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6376..6609
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(6728..6746)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6814..6918
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6919..7779
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7137..7156)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7950..8538
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8439..8458
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8692..8709
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     8801..8820
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     protein_bind    8949..8970
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        8985..9015
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9023..9039
                     /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)."
     primer_bind     9028..9050
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     9047..9063
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     9047..9063
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     9113..9132
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagtggc
     2221 ctccgcgccg ggttttggcg cctcccgcgg gcgcccccct cctcacggcg agcgctgcca
     2281 cgtcagacga agggcgcagc gagcgtcctg atccttccgc ccggacgctc aggacagcgg
     2341 cccgctgctc ataagactcg gccttagaac cccagtatca gcagaaggac attttaggac
     2401 gggacttggg tgactctagg gcactggttt tctttccaga gagcggaaca ggcgaggaaa
     2461 agtagtccct tctcggcgat tctgcggagg gatctccgtg gggcggtgaa cgccgatgat
     2521 tatataagga cgcgccgggt gtggcacagc tagttccgtc gcagccggga tttgggtcgc
     2581 ggttcttgtt tgtggatcgc tgtgatcgtc acttggcggc cgcaatgggc ccaaaaaaga
     2641 aaagaaaagt tggctacccc tacgacgtgc ccgactacgc catcgaaggc cgccatatgc
     2701 tagccgttaa aatggcttct aactttactc agttcgttct cgtcgacaat ggcggaactg
     2761 gcgacgtgac tgtcgcccca agcaacttcg ctaacgggat cgctgaatgg atcagctcta
     2821 actcgcgttc acaggcttac aaagtaacct gtagcgttcg tcagagctct gcgcagaatc
     2881 gcaaatacac catcaaagtc gaggtgccta aaggcgcctg gcgttcgtac ttaaatatgg
     2941 aactaaccat tccaattttc gccacgaatt ccgactgcga gcttattgtt aaggcaatgc
     3001 aaggtctcct aaaagatgga aacccgattc cctcagcaat cgcagcaaac tccggcatct
     3061 acgccatggc ttctaacttt actcagttcg ttctcgtcga caatggcgga actggcgacg
     3121 tgactgtcgc cccaagcaac ttcgctaacg ggatcgctga atggatcagc tctaactcgc
     3181 gttcacaggc ttacaaagta acctgtagcg ttcgtcagag ctctgcgcag aatcgcaaat
     3241 acaccatcaa agtcgaggtg cctaaaggcg cctggcgttc gtacttaaat atggaactaa
     3301 ccattccaat tttcgccacg aattccgact gcgagcttat tgttaaggca atgcaaggtc
     3361 tcctaaaaga tggaaacccg attccctcag caatcgcagc aaactccggc atctacgcgg
     3421 attctagagg tggcgggtcc ggcggtggat ccggtaccat gggccagact gggaagaaat
     3481 ctgagaaggg accggtttgt tggcggaagc gtgtaaaatc agagtacatg agactgagac
     3541 agctcaagag gttcagaaga gctgatgaag taaagactat gtttagttcc aatcgtcaga
     3601 aaattttgga aagaactgaa accttaaacc aagagtggaa gcagcggagg atacagcctg
     3661 tgcacatcat gacttctgtg agctcattgc gcgggactag ggagtgttca gtcaccagtg
     3721 acttggattt tccagcacaa gtcatcccgt taaagaccct gaatgcagtc gcctcggtgc
     3781 ctataatgta ctcttggtcg cccttacaac agaattttat ggtggaagac gaaactgttt
     3841 tacataacat tccttatatg ggggatgaag ttctggatca ggatggcact ttcattgaag
     3901 aactaataaa aaattatgat ggaaaagtgc atggtgacag agaatgtgga tttataaatg
     3961 atgaaatttt tgtggagttg gtaaatgctc ttggtcaata taatgatgat gatgatgacg
     4021 atgatggaga tgatccagat gaaagagaag aaaaacagaa agatctagag gataatcgag
     4081 atgataaaga aacttgccca cctcggaaat ttcctgctga taaaatattt gaagccattt
     4141 cctcaatgtt tccagataag ggcaccgcag aagaactgaa agaaaaatat aaagaactca
     4201 cggagcagca gctcccaggt gctctgcctc ctgaatgtac tccaaacatc gatggaccaa
     4261 atgccaaatc tgttcagagg gagcaaagct tgcattcatt tcatacgctc ttctgtcgac
     4321 gatgttttaa gtatgactgc ttcctacatc ccttccatgc aacacccaac acatataaga
     4381 ggaagaacac agaaacagct ttggacaaca agccttgtgg accacagtgt taccagcatc
     4441 tggagggagc taaggagttt gctgctgctc ttactgctga gcgtataaag acaccaccta
     4501 aacgcccagg gggccgcaga agaggaagac ttccgaataa cagtagcaga cccagcaccc
     4561 ccaccatcag tgtgctggag tcaaaggata cagacagtga cagagaagca gggactgaaa
     4621 ctgggggaga gaacaatgat aaagaagaag aagagaaaaa agatgagacg tccagctcct
     4681 ctgaagcaaa ttctcggtgt caaacaccaa taaagatgaa gccaaatatt gaacctcctg
     4741 agaatgtgga gtggagtggt gctgaagcct ccatgtttag agtcctcatt ggtacttact
     4801 acgataactt ttgtgccatt gctaggctaa ttgggaccaa aacatgtaga caggtgtatg
     4861 agtttagagt caaggagtcc agtatcatag cacctgttcc cactgaggat gtagacactc
     4921 ctccaagaaa gaagaaaagg aaacatcggt tgtgggctgc acactgcaga aagatacaac
     4981 tgaaaaagga cggctcctct aaccatgttt acaactatca accctgtgac catccacggc
     5041 agccttgtga cagttcgtgc ccttgtgtga tagcacaaaa tttttgtgaa aagttttgtc
     5101 aatgtagttc agagtgtcaa aaccgctttc ctggatgtcg gtgcaaagca caatgcaaca
     5161 ccaaacagtg tccatgctac ctggctgtcc gagagtgtga ccctgacctc tgtctcacgt
     5221 gtggagctgc tgaccattgg gacagtaaaa atgtatcctg taagaactgt agcattcagc
     5281 ggggctctaa aaagcactta ctgctggcac cgtctgatgt ggcaggctgg ggcatcttta
     5341 tcaaagatcc tgtacagaaa aatgaattca tctcagaata ctgtggggag attatttctc
     5401 aggatgaagc agacagaaga ggaaaagtgt atgacaaata catgtgcagc tttctgttca
     5461 acttgaacaa tgattttgtg gtggatgcaa cccgaaaggg caacaaaatt cgttttgcta
     5521 atcattcagt aaatccaaac tgctatgcaa aagttatgat ggttaatggt gaccacagga
     5581 taggcatctt tgctaagagg gctatccaga ctggtgaaga gttgtttttt gattacagat
     5641 acagccaggc tgatgccctg aagtatgtgg gcatcgaacg agaaatggaa atcccttaaa
     5701 tcgatagatc ctaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     5761 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     5821 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     5881 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     5941 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     6001 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     6061 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     6121 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     6181 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     6241 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     6301 cctgagatcc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     6361 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg
     6421 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     6481 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     6541 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     6601 tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
     6661 gaatatcaga gagtgagagg cccgggttaa ttaaggaaag ggctagatca ttcttgaaga
     6721 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     6781 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     6841 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     6901 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     6961 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     7021 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     7081 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     7141 tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac
     7201 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     7261 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     7321 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     7381 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     7441 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     7501 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     7561 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     7621 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     7681 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     7741 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     7801 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     7861 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     7921 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     7981 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     8041 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     8101 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     8161 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     8221 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     8281 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     8341 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     8401 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     8461 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     8521 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     8581 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     8641 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     8701 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     8761 attcattaat gcagcaagct catggctgac taattttttt tatttatgca gaggccgagg
     8821 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     8881 tttgcaaaaa gctccccgtg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     8941 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     9001 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     9061 gacatgatta cgaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     9121 tccaaactca tcaatgtatc ttatcatgtc tggatcaact ggataactca agctaaccaa
     9181 aatcatccca aacttcccac cccataccct attaccactg ccaattacct gtggtttcat
     9241 ttactctaaa cctgtgattc ctctgaatta ttttcatttt aaagaaattg tatttgttaa
     9301 atatgtacta caaacttagt agt
//
