LOCUS       sequence-382856-        8308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral construct with mCherry and Puro cassettes to express 
            sgRNA-6 targeting the promoter of human MECP2..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8308)
  AUTHORS   Qian J, Guan X, Xie B, Xu C, Niu J, Tang X, Li CH, Colecraft HM, 
            Jaenisch R, Liu XS
  TITLE     Multiplex epigenome editing of MECP2 to rescue Rett syndrome 
            neurons.
  JOURNAL   Sci Transl Med. 2023 Jan 18;15(679):eadd4666. doi: 
            10.1126/scitranslmed.add4666. Epub 2023 Jan 18.
  PUBMED    36652535
REFERENCE   2  (bases 1 to 8308)
  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 Transl 
            Med."; date: "2023-01-18"; volume: "15(679):eadd4666. doi"; pages: "
            10.1126/scitranslmed.add4666. Epub 2023 Jan 18"
COMMENT     Sequence Label: pgRNA_hMECP2_promoter_No6
FEATURES             Location/Qualifiers
     source          1..8308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        128..507
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        509..707
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             725..905
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    952..1077
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1574..1807
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1992..2036
                     /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    2334..2451
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2704..2737)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2741..2761
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     enhancer        3055..3358
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3359..3562
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3596..4192
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3596..3615)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4092..4112
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4202..4255
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      4262..4271
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4268..4978
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4413..4431)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4681..4700
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(4998..5031)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5087..5675
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5140..5160)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5558..5569)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5678..5694)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5679..5695)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6204..6384
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6446..7034)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6526..6545)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7205..7996,7997..8065))
                     /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     7828..7847
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8066..8170)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8245..8264)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc
       61 atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
      121 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
      181 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      241 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      301 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      361 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      421 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      481 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
      541 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
      601 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
      661 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg
      721 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
      781 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
      841 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
      901 tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga
      961 cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt
     1021 acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt
     1081 attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag
     1141 aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt
     1201 aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca
     1261 tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc aaccctctat
     1321 tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa
     1381 gagcaaaaca aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg
     1441 gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa
     1501 ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa
     1561 gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg
     1621 gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc
     1681 agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct
     1741 ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac
     1801 agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga
     1861 atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt
     1921 gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa
     1981 accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga
     2041 attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag
     2101 gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg
     2161 gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg
     2221 aaggaataga agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg
     2281 gatcggcact gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa
     2341 agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca
     2401 gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat
     2461 tacagggaca gcagagatcc agtttggtta gtaccgggcc cgctctagag atccgacgcg
     2521 ccatctctag gcccgcgccg gccccctcgc acggacttgt gggagaagct cggctactcc
     2581 cctgccccgg ttaatttgca tataatattt cctagtaact atagaggctt aatgtgcgat
     2641 aaaagacaga taatctgttc tttttaatac tagctacatt ttacatgata ggcttggatt
     2701 tctataactt cgtatagcat acattatacg aagttataaa cagcacaaaa ggaaactcac
     2761 cctaactgta aagtaattgt gtgttttgag actataagta tcccttggag aaccaccttg
     2821 ttggggaggg ggagggtaga gagggtttaa gagctatgct ggaaacagca tagcaagttt
     2881 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttctc
     2941 gagtactagg atccattagg cggccgcgtg gataaccgta ttaccgccat gcattagtta
     3001 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     3061 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     3121 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     3181 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     3241 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     3301 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
     3361 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
     3421 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
     3481 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
     3541 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc ccaccatgac
     3601 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtcccccggg ccgtacgcac
     3661 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgacc cggaccgcca
     3721 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     3781 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     3841 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     3901 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     3961 gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     4021 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     4081 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     4141 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg ccggatcggg
     4201 agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaccggt
     4261 cgccaccatg gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg
     4321 cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg
     4381 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc
     4441 cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt
     4501 gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg
     4561 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct
     4621 gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg
     4681 ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga
     4741 ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta
     4801 cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta
     4861 caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca
     4921 gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtaaga
     4981 attcgtcgag ggacctaata acttcgtata gcatacatta tacgaagtta tacatgttta
     5041 agggttccgg ttccactagg tacaattcga tatcaagctt atcgataatc aacctctgga
     5101 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     5161 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     5221 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     5281 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     5341 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     5401 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     5461 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     5521 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     5581 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     5641 gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgtcgac ctcgatcgag
     5701 acctagaaaa acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg
     5761 cctggctaga agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt
     5821 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     5881 gactggaagg gctaattcac tcccaacgaa gacaagatat ccttgatctg tggatctacc
     5941 acacacaagg ctacttccct gattggcaga actacacacc agggccaggg atcagatatc
     6001 cactgacctt tggatggtgc tacaagctag taccagttga gcaagagaag gtagaagaag
     6061 ccaatgaagg agagaacacc cgcttgttac accctgtgag cctgcatggg atggatgacc
     6121 cggagagaga agtattagag tggaggtttg acagccgcct agcatttcat cacatggccc
     6181 gagagctgca tccggactgt actgggtctc tctggttaga ccagatctga gcctgggagc
     6241 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     6301 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     6361 agtcagtgtg gaaaatctct agcagcatgt gagcaaaagg ccagcaaaag gccaggaacc
     6421 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     6481 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     6541 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     6601 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     6661 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6721 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6781 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6841 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     6901 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6961 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     7021 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     7081 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     7141 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     7201 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     7261 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     7321 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     7381 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     7441 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     7501 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     7561 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     7621 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     7681 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7741 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7801 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7861 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7921 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7981 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     8041 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     8101 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     8161 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     8221 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     8281 tctgctccct gcttgtgtgt tggaggtc
//
