LOCUS       sequence-382873-        8308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral construct with mCherry and Puro cassettes to express 
            sgRNA-8 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_No8
FEATURES             Location/Qualifiers
     source          1..8308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        148..527
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        529..727
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             745..925
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    972..1097
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1594..1827
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2012..2056
                     /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    2354..2471
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2724..2757)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2761..2781
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     enhancer        3075..3378
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3379..3582
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3616..4212
                     /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(3616..3635)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4112..4132
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4222..4275
                     /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      4282..4291
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4288..4998
                     /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(4433..4451)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4701..4720
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(5018..5051)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5107..5695
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5160..5180)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5578..5589)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5698..5714)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5699..5715)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6224..6404
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6466..7054)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6546..6565)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7225..8016,8017..8085))
                     /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     7848..7867
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8086..8190)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8265..8284)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
       61 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
      121 gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat
      181 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      241 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      301 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      361 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      421 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
      481 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
      541 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
      601 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
      661 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
      721 taagcagcgc gttttgcctg tactgggtct ctctggttag accagatctg agcctgggag
      781 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
      841 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
      901 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
      961 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
     1021 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
     1081 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
     1141 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
     1201 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
     1261 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
     1321 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     1381 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
     1441 gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata
     1501 taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag
     1561 agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg
     1621 agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt
     1681 attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca
     1741 tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga
     1801 aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg
     1861 caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa
     1921 tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct taatacactc
     1981 cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga
     2041 taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt
     2101 attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat
     2161 agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc
     2221 gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag
     2281 atccattcga ttagtgaacg gatcggcact gcgtgcgcca attctgcaga caaatggcag
     2341 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
     2401 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa
     2461 ttcaaaattt tcgggtttat tacagggaca gcagagatcc agtttggtta gtaccgggcc
     2521 cgctctagag atccgacgcg ccatctctag gcccgcgccg gccccctcgc acggacttgt
     2581 gggagaagct cggctactcc cctgccccgg ttaatttgca tataatattt cctagtaact
     2641 atagaggctt aatgtgcgat aaaagacaga taatctgttc tttttaatac tagctacatt
     2701 ttacatgata ggcttggatt tctataactt cgtatagcat acattatacg aagttataaa
     2761 cagcacaaaa ggaaactcac cctaactgta aagtaattgt gtgttttgag actataagta
     2821 tcccttggag aaccaccttg ttggtgagag ctcaggagcc cttggtttaa gagctatgct
     2881 ggaaacagca tagcaagttt aaataaggct agtccgttat caacttgaaa aagtggcacc
     2941 gagtcggtgc tttttttctc gagtactagg atccattagg cggccgcgtg gataaccgta
     3001 ttaccgccat gcattagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
     3061 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
     3121 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
     3181 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
     3241 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     3301 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     3361 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
     3421 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     3481 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
     3541 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag
     3601 atccgctagc ccaccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     3661 gtcccccggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     3721 accgtcgacc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     3781 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     3841 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     3901 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     3961 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag
     4021 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     4081 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     4141 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     4201 aagcccggtg ccggatcggg agagggcaga ggaagtctgc taacatgcgg tgacgtcgag
     4261 gagaatcctg gcccaccggt cgccaccatg gtgagcaagg gcgaggagga taacatggcc
     4321 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
     4381 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     4441 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
     4501 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     4561 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     4621 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     4681 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     4741 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     4801 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     4861 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     4921 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     4981 gacgagctgt acaagtaaga attcgtcgag ggacctaata acttcgtata gcatacatta
     5041 tacgaagtta tacatgttta agggttccgg ttccactagg tacaattcga tatcaagctt
     5101 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     5161 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     5221 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     5281 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     5341 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     5401 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     5461 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     5521 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     5581 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     5641 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     5701 taccgtcgac ctcgatcgag acctagaaaa acatggagca atcacaagta gcaatacagc
     5761 agctaccaat gctgattgtg cctggctaga agcacaagag gaggaggagg tgggttttcc
     5821 agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca
     5881 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatat
     5941 ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga actacacacc
     6001 agggccaggg atcagatatc cactgacctt tggatggtgc tacaagctag taccagttga
     6061 gcaagagaag gtagaagaag ccaatgaagg agagaacacc cgcttgttac accctgtgag
     6121 cctgcatggg atggatgacc cggagagaga agtattagag tggaggtttg acagccgcct
     6181 agcatttcat cacatggccc gagagctgca tccggactgt actgggtctc tctggttaga
     6241 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     6301 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     6361 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagcatgt gagcaaaagg
     6421 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     6481 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     6541 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     6601 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     6661 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     6721 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     6781 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     6841 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     6901 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     6961 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     7021 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     7081 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     7141 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     7201 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     7261 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     7321 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     7381 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     7441 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     7501 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     7561 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     7621 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     7681 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     7741 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     7801 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     7861 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     7921 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     7981 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     8041 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     8101 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     8161 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     8221 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat
     8281 gccgcatagt taagccagta tctgctcc
//
