LOCUS       sequence-244974-        7274 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MCP (MS2 Coat Protein) fusion to KRAB in mammalian cells, 
            lentiviral backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7274)
  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 7274)
  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: "Epigenetics
            Chromatin."; date: "2019-05-3"; volume: "12(1):26. doi"; pages: " 
            10.1186/s13072-019-0275-8"
COMMENT     Sequence Label: NLS-HA-2xMCP-KRAB
FEATURES             Location/Qualifiers
     source          1..7274
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     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    3664..4252
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3717..3737)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4135..4146)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4327..4560
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(4679..4697)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4765..4869
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4870..4938,4939..5730)
                     /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(5088..5107)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5901..6489
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6390..6409
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6643..6660
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6900..6921
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6936..6941,6942..6959,6960..6966)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6974..6990
                     /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     6979..7001
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6998..7014
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6998..7014
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7064..7083
                     /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 attctagaat ggtgaccttc aaggatgtat ttgtggactt caccagggag gagtggaagc
     3481 tgctggacac tgctcagcag atcgtgtaca gaaatgtgat gctggagaac tataagaacc
     3541 tggtttcctt gggttatcag cttactaagc cagatgtgat cctccggttg gagaagggag
     3601 aagagccctg gctggtggag agagaaattc accaagagac ccatccttaa atcgatagat
     3661 cctaatcaac ctctggatta caaaatttgt aaaagattga ctggtattct taactatgtt
     3721 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     3781 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     3841 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     3901 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     3961 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     4021 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     4081 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     4141 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     4201 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcctgagatc
     4261 ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg
     4321 ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg
     4381 ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac
     4441 tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt
     4501 gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca
     4561 gtagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag
     4621 agagtgagag gcccgggtta attaaggaaa gggctagatc attcttgaag acgaaagggc
     4681 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     4741 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     4801 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     4861 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     4921 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     4981 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5041 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5101 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5161 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5221 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5281 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     5341 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5401 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     5461 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     5521 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     5581 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     5641 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     5701 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     5761 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     5821 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     5881 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     5941 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     6001 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     6061 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     6121 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     6181 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     6241 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     6301 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     6361 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     6421 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     6481 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     6541 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     6601 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     6661 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     6721 tgcagcaagc tcatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     6781 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     6841 agctccccgt ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     6901 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt
     6961 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgacatgatt
     7021 acgaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc
     7081 atcaatgtat cttatcatgt ctggatcaac tggataactc aagctaacca aaatcatccc
     7141 aaacttccca ccccataccc tattaccact gccaattacc tgtggtttca tttactctaa
     7201 acctgtgatt cctctgaatt attttcattt taaagaaatt gtatttgtta aatatgtact
     7261 acaaacttag tagt
//
